Real-Time PCR

CFX Maestro™ Software

User Guide
Version 1.1
CFX Maestro™ Software
User Guide
Version 1.1
Bio-Rad Technical Support Department
The Bio-Rad Technical Support department in the U.S. is open Monday through Friday, 5:00 AM to
5:00 PM, Pacific Time.
Phone: 1-800-424-6723, option 2
Email: [email protected] (U.S./Canada Only)

For technical assistance outside the U.S. and Canada, contact your local technical support office or
click the Contact us link at www.bio-rad.com.

Notice
No part of this publication may be reproduced or transmitted in any form or by any means,
electronic or mechanical, including photocopy, recording, or any information storage or retrieval
system, without permission in writing from Bio-Rad Laboratories, Inc.
Bio-Rad reserves the right to modify its products and services at any time. This guide is subject to
change without notice. Although prepared to ensure accuracy, Bio-Rad assumes no liability for
errors or omissions, or for any damage resulting from the application or use of this information.
The following are trademarks of Bio-Rad Laboratories, Inc.: Bio-Rad, CFX Maestro, CFX384,
CFX384 Touch, CFX96 Touch, CFX Connect, and C1000 Touch and iProof and iTaq.
Adobe, Acrobat, and Reader are trademarks of Adobe Systems Incorporated. Cy is a trademark of
GE Healthcare Group companies. CAL Fluor and Quasar are trademarks of Biosearch
Technologies, Inc. SYBR and Texas Red are trademarks of Life Technologies Corporation. Bio-
Rad Laboratories, Inc. is licensed by Life Technologies Corporation to sell reagents containing
SYBR Green for use in real-time PCR, for research purposes only. Excel, Microsoft, PowerPoint,
and Windows are trademarks of Microsoft Corporation. Gmail is a trademark of Google, Inc. Yahoo!
is a trademark of Yahoo! Inc. Pentium is a trademark of Intel Corporation.
EvaGreen is a trademark of Biotium, Inc. FAM, ROX, and VIC are trademarks of Applera
Corporation. Bio-Rad Laboratories, Inc. is licensed by Applera Corporation to sell reagents
containing EvaGreen dye for use in real-time PCR, for research purposes only.
Bio-Rad thermal cyclers and real-time systems are covered by one or more of the following U.S.
patents or their foreign counterparts owned by Eppendorf AG: U.S. Patent Numbers 6,767,512 and
7,074,367.
Hard-Shell plates are covered by one or more of the following U.S. patents or their foreign
counterparts owned by Eppendorf AG: U.S. Patent Numbers 7,347,977; 6,340,589; and 6,528,302.

Copyright © 2017 by Bio-Rad Laboratories, Inc. All rights reserved.

Table of Contents
Safety and Regulatory Compliance 11
Safety Warning Labels 11
Safe Use Specifications and Compliance 12
Regulatory Compliance 12
Hazards 13
Biohazards 13
Chemical Hazards 15
Explosive or Flammability Hazards 15
Electrical Hazards 15
Transport 15
Battery 15
Disposal 16
Warranty 16

Chapter 1 Introduction 1
Main Features of CFX Maestro Software 2
Finding Out More 2

Chapter 2 Installing CFX Maestro Software 3

System Requirements 4
Installing CFX Maestro Software 5
Detecting Connected Instruments 6
Software Files 7

Chapter 3 The Workspace 9

The Home Window 10
The Startup Wizard 11
The Protocol Editor Window 12
The Plate Editor Window 13
The Data Analysis Window 14

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Table of Contents

Chapter 4 The Home Window 15

The Home Window 16
File Menu Commands 17
View Menu Commands 17
User Menu Commands 18
Run Menu Commands 18
Tools Menu Commands 19
Help Menu Commands 19
Toolbar Commands 20
The Startup Wizard 21
Status Bar 21
Detected Instruments Pane 22
Viewing the Properties of an Instrument 26
Before You Begin 28
Performing Software and Instrument Updates 28
Setting User Preferences 29
Creating a Reaction Master Mix 43
Calibrating New Dyes 46

Chapter 5 Creating Protocols 49

Protocol Editor Window 50
File Menu Commands 50
Settings Menu Command 51
Tools Menu Commands 51
Toolbar Commands 51
Protocol Editing Controls 52
Creating a Protocol in the Protocol Editor 54
Opening a New Protocol File in the Protocol Editor 54
Opening an Existing Protocol in the Protocol Editor 55
Setting Up a New Protocol 56
Adding Steps to a Protocol 58
Inserting a Gradient Step 59
Inserting a GOTO Step 60
Inserting a Melt Curve Step 60
Adding or Removing a Plate Read Step 62

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 Table of Contents

Changing Step Options 62

Deleting a Step 63
Copying, Exporting, or Printing a Protocol 63
Creating a Protocol with the Protocol AutoWriter 64
Using the Ta Calculator 66
About the Ta Calculator 66

Chapter 6 Preparing Plates 71

Plate Editor Window 72
File Menu Commands 72
Edit Menu Commands 73
Settings Menu Commands 73
Editing Tools Menu Commands 73
Toolbar Commands 74
Creating a Plate File Using the Plate Editor 75
Opening a New Plate File in the Plate Editor 75
Opening an Existing Plate File in the Plate Editor 77
Setting Up a New Plate File 78
Assigning Optional Parameters to the Plate File 84
Assigning a Target to Wells 84
Assigning a Sample Name to Wells 86
Assigning Biological Groups to Wells 88
Assigning Technical Replicate Numbers to Wells 90
Assigning a Dilution Series to Standard Sample Types 91
Copying Well Contents into Another Well 93
Adding a Note to a Well 93
Clearing Wells of All Content 94
Changing Experiment Settings 95
Creating Well Groups 97
Changing Trace Styles 99
Viewing the Plate in Spreadsheet Format 100
Creating a Plate Layout Using the Plate Setup Wizard 102
Using the Plate Setup Wizard 102

Chapter 7 Running Experiments 105

The Run Setup Window 106

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Table of Contents

Accessing the Run Setup Window 107

Protocol Tab 108
Plate Tab 110
Start Run Tab 112
Running an Experiment 113
Run Details Dialog Box 115
Run Status Tab 115
Real-time Status Tab 117
Time Status Tab 119
Performing PrimePCR Experiments 120
Transferring Stand-Alone Data for Analysis 122
Transferring Data Through CFX Maestro Software 122
Transferring Data Through Email 122
Transferring Data Using a USB Drive 123
Creating a Data File 124

Chapter 8 Data Analysis Overview 125

Data Analysis Window 125
Data Analysis Toolbar 126
Data Analysis Menu Bar 127
Tab Details 129
Step Number Selector 130
Viewing Well Groups in Data Analysis 130
Changing Well Contents after a Run 131
Data Analysis Settings 132
Adjusting the Threshold 132
Baseline Settings 132
Analysis Mode 133
Cycles to Analyze 134
Well Selector 135
Well Selector Right-Click Menu Items 136
Temporarily Excluding Wells from Analysis 137
Charts 138
Chart Tools 138
Magnifying an Area in the Chart 146

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 Table of Contents

Copying Charts into a Microsoft File 146

Common Right-Click Menu Items for Charts 146
Spreadsheets 147
Common Right-Click Menu Items for Spreadsheets 147
Export 149
Exporting All Data Sheets to Excel 149
Exporting RDML Files 149
Creating a Custom Export File 150
Exporting to a LIMS Folder 151

Chapter 9 Data Analysis Details 153

Quantification Tab 154
Fluorophore Options 154
Trace Styles Dialog Box 155
Log Scale Option 156
Standard Curve Chart 157
Amplification Chart Menu Options 158
Quantification Tab Spreadsheet 158
Quantification Data Tab 160
Results Spreadsheet 160
Standard Curve Results Spreadsheet 162
Plate Spreadsheet 163
RFU Spreadsheet 164
Melt Curve Tab 165
Adjusting Melt Curve Data 166
Melt Curve Data Tab 167
Melt Peaks Spreadsheet 167
Plate Spreadsheet 168
RFU Spreadsheet 169
-d(RFU)/dT Spreadsheet 170
End Point Tab 171
Results Data 172
Adjusting the End Point Data Analysis 173
RFU Spreadsheet for End Point Analysis 173
Allelic Discrimination Tab 174

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Table of Contents

Adjusting Data for Allelic Discrimination 175

Chart Menu Options 176
Allelic Discrimination Spreadsheet 176
Custom Data View Tab 177
Creating a Custom Data View 178
QC Tab 179
Changing QC Criteria 179
Excluding Wells That Fail QC 180
Run Information Tab 181
Data Analysis Reports 182
Data Analysis Report Categories 183
Creating a Data Analysis Report 186
Creating Well Group Reports 187

Chapter 10 Gene Expression Analysis 189

Plate Setup for Gene Expression Analysis 189
Guided Plate Setup 190
Gene Expression Charts 191
Graphing 193
Changing and Annotating the Chart View 195
Adjusting Gene Expression Data 201
Experiment Settings 204
Right-Click Menu Options 205
Data Spreadsheet 206
Show Details Option 208
Clustergram 211
Settings 211
Right-Click Menu Options 211
Data Spreadsheet 211
Scatter Plot 212
Settings 212
Right-Click Menu Options 212
Data Spreadsheet 213
Volcano Plot 214
Settings 214

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 Table of Contents

Right-Click Menu Options 214

Data Spreadsheet 215
Heat Map 216
Settings 216
Right-Click Menu Options 216
Data Spreadsheet 216
ANOVA 217
Settings 217
Results Table 218
Right-Click Menu Options 220
Gene Study 221
Inter-Run Calibration 221
Gene Study Dialog Box 222
Study Setup Tab 222
Preparing a Gene Study 223
Study Analysis Tab 224
Gene Study Reports 225
Creating a Gene Study Report 227

Appendix A Data Analysis Calculations 229

P-Value 229
Differences in P-Value Results 230
Reaction Efficiency 231
Relative Quantity 231
Relative Quantity When a Control Is Selected 232
Standard Deviation of Relative Quantity 232
Efficiency Corrected Cq (CqE) 233
Mean Efficiency Corrected Cq (MCqE) 233
Normalized Expression 233
Expression and Relative Quantity for Biological Groups 234
Normalized Expression When a Control Is Selected 234
Standard Deviation for the Normalized Expression 234
Normalized Expression Scaled to Highest Expression Level 235
Normalized Expression Scaled to Lowest Expression Level 235
Normalized Expression Scaled to Average Expression Level 236

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Table of Contents

Standard Deviation for the Scaled Normalized Expression 237

Error Bars for Standard Deviation(lg) and Standard Error of the Mean (lg) 238
Fold Change 239
Corrected Values Formulas 240
Confidence Interval Calculation for Biological Group Analysis 241
Box and Whisker Chart Calculations 241

Appendix B Managing CFX Maestro Users and Roles 243

Managing Users 243
Adding and Removing Users 243
Managing the Rights of Roles 245
Logging In to CFX Maestro Software 246
Changing Users 246
Changing User Passwords 247
Viewing Your Role and Rights 247

Appendix C LIMS Integration 249

Creating LIMS-Compatible Data Files 249
Setting Up LIMS Folder and Data Export Options 249
Creating a LIMS Protocol 250
Creating a LIMS File 251
Starting a LIMS Run 254
Exporting Data to a LIMS 255

Appendix D Troubleshooting CFX Maestro Software Connection Issues 257

Application Log 257
Troubleshooting 258
Power Failure 258
Recovering Data Files to a USB Flash Drive 260
Retrieving Files to the CFX Maestro Computer 260
Installing CFX Maestro Software Manually 260
Reinstalling the Drivers 261

Appendix E References 263

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Safety and Regulatory Compliance
For safe operation of the C1000™ Touch system, CFX96™ Touch system, CFX96 Touch Deep
Well system, CFX Connect™ system or CFX385™ Touch system, we strongly recommend that you
follow the safety specifications listed in this section and throughout this manual.

Safety Warning Labels

Warning labels posted on the instrument and in this manual warn you about sources of injury or
harm. Table 1 defines each safety warning label.

Table 1. Meaning of safety warning labels

Icon Meaning

Warning about risk of harm to body or equipment

Operating the CFX system before reading this manual can constitute a personal injury
hazard. For safe use, do not operate this instrument in any manner unspecified in this
manual. Only qualified laboratory personnel trained in the safe use of electrical
equipment should operate this instrument. Always handle all components of the system
with care and with clean, dry hands.

Warning about handling biohazardous materials

When handling biohazardous samples, adhere to the recommended precautions and
guidelines and comply with any local guidelines specific to your laboratory and location.

Warning about risk of burning

A thermal cycler generates enough heat to cause serious burns. Wear safety goggles or
other eye protection at all times during operation. Always allow the sample block to
return to idle temperature before opening the lid and removing samples. Always allow
maximum clearance to avoid accidental skin burns.

Warning about risk of explosion

The sample blocks can become hot enough during the course of normal operation to
cause liquids to boil and explode.

Note: For information about the C1000 Touch and CFX Connect thermal cyclers, refer to their
respective instrument guides.

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Safety and Regulatory Compliance

Safe Use Specifications and Compliance

Table 2 lists the safe use specifications for Bio-Rad’s CFX real-time PCR detection systems. The
supplied shielded cables must be used with these instruments to ensure compliance with the
Class A FCC limits.

Table 2. Conditions for safe use

Usage Aspect Conditions for Safe Use

Rated input power 100–240 VAC, 50–60 Hz, 850 W max

Overvoltage category II

Fuses 10 A, 250 V, 5 x 20 mm, fast blow (qty. 2)

Environment Indoor use only

Usage temperature 15–31°C

Storage temperature –20 to 60°C

Relative humidity Up to 80% (noncondensing)

Altitude Up to 2,000 meters above sea level

Pollution degree 2

Regulatory Compliance
The CFX real-time PCR detection system has been tested and found to be in compliance with all
applicable requirements of the following safety and electromagnetic standards:

n IEC 61010-1:2001 (2nd ed.), EN61010-1:2001 (2nd ed). Electrical equipment for
measurement, control, and laboratory use - Part 1: General requirements

n IEC 61010-2-010:2005, EN61010-2-010:2003. Safety requirements for electrical equipment

for measurement, control, and laboratory use. Part 2-010: Particular requirements for
laboratory equipment for the heating of materials

n IEC 61010-2-081:2001+A1, EN61010-2-081:2002+A1. Safety requirements for electrical

equipment for measurement, control, and laboratory use. Part 2-081: Particular requirements
for automatic and semi-automatic laboratory equipment for analysis and other purposes
(includes Amendment 1)

n EN 61326-1:2006 (Class A). Electrical equipment for measurement, control, and laboratory
use. EMC requirements, Part 1: General requirements

12 | CFX Maestro Software

 Hazards

Important: This equipment generates, uses, and can radiate radiofrequency energy and, if not
installed and used in accordance with the instruction manual, may cause harmful interference
to radio communications. Operation of the systems in a residential area is likely to cause
harmful interference, in which case users will be required to correct the interference at their
own expense.

Hazards
The CFX real-time PCR detection system is designed to operate safely when used in the manner
prescribed by the manufacturer. If the CFX real-time PCR detection system or any of its associated
components is used in a manner not specified by the manufacturer, the inherent protection
provided by the instrument may be impaired. Bio-Rad Laboratories, Inc. is not liable for any injury
or damage caused by the use of this equipment in any unspecified manner, or by modifications to
the instrument not performed by Bio-Rad or an authorized agent. Service of the CFX real-time PCR
detection system should be performed only by trained Bio-Rad personnel.

Biohazards
The CFX real-time PCR detection system is a laboratory product. However, if biohazardous
samples are present, adhere to the following guidelines and comply with any local guidelines
specific to your laboratory and location.

Note: No biohazardous substances are exhausted during normal operations of this

instrument.

General Precautions

n Always wear laboratory coat, laboratory gloves, and safety glasses with side shields or
goggles.

n Keep your hands away from your mouth, nose, and eyes.

n Completely protect any cut or abrasion before working with potentially infectious materials.

n Wash your hands thoroughly with soap and water after working with any potentially infectious
material before leaving the laboratory.

n Remove wristwatches and jewelry before working at the bench.

n Store all infectious or potentially infectious material in unbreakable leak-proof containers.

n Before leaving the laboratory, remove protective clothing.

n Do not use a gloved hand to write, answer the telephone, turn on a light switch, or touch
anything that other people may touch without gloves.

User Guide | 13
Safety and Regulatory Compliance

n Change gloves frequently. Remove gloves immediately when they are visibly contaminated.

n Do not expose materials that cannot be properly decontaminated to potentially infectious

material.

n Upon completion of an operation involving biohazardous material, decontaminate the work

area with an appropriate disinfectant (for example, a 1:10 dilution of household bleach).

Surface Decontamination

WARNING! To prevent electrical shock, always turn off and unplug the instrument
prior to performing decontamination procedures.

The following areas can be cleaned with any hospital-grade bactericide, virucide, or fungicide
disinfectant:

n Outer lid and chassis

n Inner reaction block surface and reaction block wells

n Control panel and display

To prepare and apply the disinfectant, refer to the instructions provided by the product
manufacturer. Always rinse the reaction block and reaction block wells several times with water
after applying a disinfectant. Thoroughly dry the reaction block and reaction block wells after
rinsing with water.

Important: Do not use abrasive or corrosive detergents or strong alkaline solutions. These
agents can scratch surfaces and damage the reaction block, resulting in loss of precise
thermal control.

Disposal of Biohazardous Material

Dispose of the following potentially contaminated materials in accordance with laboratory local,
regional, and national regulations:

n Clinical samples

n Reagents

n Used reaction vessels or other consumables that may be contaminated

14 | CFX Maestro Software

 Hazards

Chemical Hazards
The CFX real-time PCR detection system contains no potentially hazardous chemical materials.

Explosive or Flammability Hazards

The CFX real-time PCR detection system poses no uncommon hazard related to flammability or
explosion when used in a proper manner as specified by Bio-Rad Laboratories.

Electrical Hazards
The CFX real-time PCR detection system poses no uncommon electrical hazard to operators if
installed and operated properly without physical modification and connected to a power source of
proper specification.

Transport
Before moving or shipping the CFX real-time PCR detection system or its optical reaction module,
decontamination procedures must be performed. Always move or ship the CFX real-time PCR
detection system and optical reaction modules in separate containers with the supplied packaging
materials, which will protect the instrument from damage. If appropriate containers cannot be
found, contact your local Bio-Rad office.

Battery
The C1000 Touch thermal cycler uses a 3 V lithium-metal coin cell battery and a 4.8 V nickel-metal
hydride rechargeable battery pack to maintain time settings and run data in the event of AC power
loss. If the time and/or run data do not remain set after the unit is turned off, it may be an indication
that the batteries are getting weak. If this occurs, contact Bio-Rad Technical Support for assistance.

Do not attempt to change the batteries. Contact Bio-Rad Technical Support.

For the State of California, USA only

n Perchlorate material – special handling may apply, see

www.dtsc.ca.gov/hazardouswaste/perchlorate

n Perchlorate material – lithium battery contains perchlorate

User Guide | 15
Safety and Regulatory Compliance

Disposal
The CFX real-time PCR detection system contains electrical materials; they should be disposed of
as unsorted waste and must be collected separately, according to European Union Directive
200296CE on waste and electronic equipment — WEEE Directive. Before disposal, contact your
local Bio-Rad representative for country-specific instructions.

Warranty
The CFX real-time PCR detection system and its associated accessories are covered by a
standard Bio-Rad warranty. Contact your local Bio-Rad office for the details of the warranty.

16 | CFX Maestro Software

Chapter 1 Introduction
Bio-Rad’s high-performance PCR amplification systems feature the latest technological advances,
providing greater accuracy and reproducibility in nucleic acid amplification for genomic
experiments. Bio-Rad’s PCR product line includes real-time and conventional PCR systems:

Real-Time PCR Systems

n CFX96 Touch™

n CFX96 Touch Deep Well

n CFX384 Touch™

n CFX Connect™

n CFX Automation System II

Conventional PCR Systems

n C1000™

n C1000 Touch™

n S1000™

Bio-Rad’s CFX Maestro™ software is compatible with all CFX instruments and features optimized
run files for Bio-Rad’s PrimePCR™ primer and probe assays.

Using CFX Maestro software you can interpret complex data and craft powerful studies for genetic
analysis. With just a few clicks, you can set up studies and make sense of your gene expression
study with tools such as t-tests, one-way ANOVA, PrimePCR controls analysis, and the reference
gene selector tool. Then, you can prepare your results for publications and posters with CFX
Maestro’s highly customizable data visualization and annotation tools.

User Guide | 1
Chapter 1 Introduction

Main Features of CFX Maestro Software

With CFX Maestro software you can do the following:

n Quickly and easily set up real-time PCR experiments using the Startup Wizard.

n Analyze data using bar charts, clustergrams, scatter plots, heat maps, or volcano plots to
quickly interpret and understand your results.

n Customize your data representation and export high resolution graphs for publication and
report generation.

n Determine RNA quality and troubleshoot experiments with PrimePCR analysis controls.

n Select the appropriate reference gene and analyze its stability with the Reference Gene
selection tool.

n Perform statistical analysis including one-way ANOVA in gene expression analysis.

n Automatically generate a PDF report with pass/fail results for each criterion to help assess
your instrument’s performance.

This user guide explains these features and how to use them.

Finding Out More

After installing the Bio-Rad real-time PCR instrument and CFX Maestro software, you can access
this guide as well as detailed CFX Maestro software help topics from the Help menu in any view.

Tip: Click the Bio-Rad logo in the upper right corner of any CFX Maestro software window to
launch Bio-Rad’s website. This site includes links to technical notes, manuals, videos, product
information, and technical support. This site also provides many technical resources on a wide
variety of methods and applications related to PCR, real-time PCR, and gene expression.

2 | CFX Maestro Software

Chapter 2 Installing CFX Maestro Software
This chapter explains how to install CFX Maestro™ software. For information about setting up
Bio-Rad's supported real-time PCR instruments, see the appropriate guide.

CFX Maestro is required to analyze real-time PCR data from the CFX96 Touch™, CFX96 Touch
Deep Well, CFX Connect™, or CFX384 Touch™ systems. You can also use this software to control
these systems in software-controlled mode.

The CFX96™, CFX96 Deep Well, CFX Connect, and CFX384™ optical reaction module shipping
package includes the optical reaction module and a USB cable.

Remove the packing materials and store them for future use. If an item is missing or damaged,
contact your local Bio-Rad office.

User Guide | 3
Chapter 2 Installing CFX Maestro Software

System Requirements
Table 3 lists the minimum and recommended system requirements for the computer running the
CFX Maestro software (known as the CFX Maestro computer).

Table 3. Computer requirements for CFX Maestro software

System Minimum Recommended

Operating system Microsoft Windows 7 SP1 Any of the following:
Pro n Microsoft Windows 7 SP2 Pro (32- and 64-bit)
n Microsoft Windows 10 Pro (64-bit only)
n Microsoft Windows 10 Enterprise (64-bit only)

Important: Secure Boot must be disabled on both Microsoft Windows 10 Pro and Enterprise.

Ports 2 USB 2.0 High-speed ports 2 USB 2.0 High-speed ports

Hard disk space 128 GB 128 GB

Processor speed 2.4 GHz, Dual Core 2.4 GHz, Quad Core

RAM 4 GB RAM 8 GB RAM

Screen resolution 1024 x 768 with true-color 1280 x 1024 with true-color mode
mode

PDF reader Adobe PDF Reader or Windows PDF Reader

from one of the supported Microsoft Office
Suites:
n 2007
n 2010
n 2013

Localization Supported Microsoft Supported Microsoft Windows 32- and 64-bit OS

Windows 32-and 64-bit OS in English, Chinese, and Russian
in English, Chinese, and
Russian

Note: If you plan to run CFX Automation Control software on the same computer as CFX
Maestro, set the screen resolution to 1280 x 1024 with true-color mode.

Note: The MiniOpticon™ system is supported only on computers running the Microsoft
Windows 7 operating system. CFX Maestro does not recognize the MiniOpticon system on
computers running either version of the Microsoft Windows 10 operating system.

4 | CFX Maestro Software

 Installing CFX Maestro Software

Installing CFX Maestro Software

Important: You must disconnect any connected instruments from the CFX Maestro computer
before you install or upgrade the software. You do not need to turn off the instrument during
the software installation. Ensure that you have saved all runs and that no experiments are
running.

Note: If you are installing CFX Maestro software on either version of Windows 10, verify that
Secure Boot is disabled before beginning the installation procedure.

To install CFX Maestro software

1. If necessary, disconnect any connected instruments from the computer.

Locate and disconnect the instrument’s USB cable on the CFX Maestro computer. The end
inserted in the instrument can remain in place.

2. Log in to the CFX Maestro computer with administrative privileges.

3. Insert the CFX Maestro software USB drive into the computer’s USB port.

4. In Windows Explorer, navigate to and open the CFX Maestro software USB drive.

The USB drive contains the Release Notes and the following folders:

n CFX

n Drivers

n Firmware

n Quick Start

Along with other files, the CFX folder contains the CFX Maestro software installer
(CFXMaestroSetup1.1.exe) and the CFX documentation installer
(CFX_Documentation.exe).

5. Open the CFX folder and double-click CFXMaestroSetup1.1.exe to start the CFX Maestro
software installer.

6. Follow the on-screen installation instructions.

When completed, the Bio-Rad CFX Maestro software splash screen appears on the computer
screen and the Bio-Rad CFX Maestro icon appears on the desktop.

7. Double-click CFX_Documentation.exe to install CFX Maestro software documentation.

8. After the installation completes, you can safely eject the software USB drive.

User Guide | 5
Chapter 2 Installing CFX Maestro Software

Detecting Connected Instruments

During installation, the CFX Maestro software installer automatically installs the instrument drivers
onto the CFX Maestro computer. CFX Maestro detects connected instruments when you start the
software.

To detect connected instruments

1. If you have not yet done so, insert the square (male) end of the supplied USB Type B cable
into the USB Type B port located on the back of the instrument’s base.

2. Insert the other (port) end into a USB port on the CFX Maestro computer.

3. If the instrument is not already running, press the power switch on the back of the instrument to
turn it on.

4. Start CFX Maestro.

The software automatically detects the connected instrument and displays its name in the Detected
Instruments pane on the Home window.

Note: If the instrument does not appear in the Detected Instruments pane, verify that the USB
cable is properly installed. To reinstall drivers, on the Home window in CFX Maestro select
Tools > Reinstall Instrument Drivers.

6 | CFX Maestro Software

 Software Files

Software Files
Table 4 lists the CFX Maestro software file types.

Table 4. CFX Maestro software file types

File Type Extension Details

Protocol .prcl Contains protocol setup details to perform a PCR run.

Plate .pltd Contains plate setup details to perform a PCR run.

Data .pcrd Contains the results of an experiment run and PCR

analysis.

PrimePCR™ run .csv Contains protocol and plate layout for PrimePCR plates.

Gene study .mgxd Contains results of multiple PCR runs and gene
expression analyses.

Stand-alone pre-data .zpcr Contains fluorescence readings from stand-alone

file operation that are converted into a data file.

LIMS .plrn Contains plate setup and protocol information required to

conduct a LIMS compatible run.

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Chapter 2 Installing CFX Maestro Software

8 | CFX Maestro Software

Chapter 3 The Workspace
CFX Maestro™ software provides an interface for setting up plates, developing PCR protocols,
running them on CFX instruments, and analyzing data from PCR runs.

CFX Maestro software presents five primary workspaces:

n The Home window

n The Startup Wizard

n The Protocol Editor window

n The Plate Editor window

n The Data Analysis window

Each workspace is shown and briefly described in this chapter.

User Guide | 9
Chapter 3 The Workspace

The Home Window

CFX Maestro software opens to the Home window and displays the Startup Wizard, from which you
can set up an experiment, perform or repeat a run, or analyze an existing run. From the Home
window you can also view application and instrument logs, create and manage users, and access
multiple useful tools. For more information, see Chapter 4, The Home Window.

10 | CFX Maestro Software

 The Startup Wizard

The Startup Wizard

Use the Startup Wizard to quickly set up and run user-defined experiments or select and run a
PrimePCR™ experiment. You can also use this wizard to repeat a run or analyze run data.

User Guide | 11
Chapter 3 The Workspace

The Protocol Editor Window

In the Protocol Editor you can create, open, review, and edit a protocol. You can also modify the lid
temperature for the open protocol. Protocol Editor functionality is detailed in Chapter 5, Creating
Protocols.

12 | CFX Maestro Software

 The Plate Editor Window

The Plate Editor Window

In the Plate Editor you can create, open, review, and edit a plate. Plate Editor functionality is
detailed in Chapter 6, Preparing Plates.

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Chapter 3 The Workspace

The Data Analysis Window

In the Data Analysis window you can view and compare run data, perform statistical analyses,
export data, and create publication-ready reports. Data Analysis functionality is detailed in Chapter
8, Data Analysis Overview. See also Chapter 9, Data Analysis Details.

14 | CFX Maestro Software

Chapter 4 The Home Window
CFX Maestro™ software provides an interface for developing PCR protocols, running them on CFX
instruments, and analyzing PCR run data.

This chapter introduces the CFX Maestro software and describes the features accessible from the
Home window.

User Guide | 15
Chapter 4 The Home Window

The Home Window

CFX Maestro opens to the Home window and displays the Startup Wizard, from which you can set
up a run, perform or repeat a run, or analyze an existing run. From the Home window you can also
view application and instrument logs, create and manage users, and access multiple useful tools.

LEGEND

1. The software title bar displays the name of the software and the logged in user.

2. The menu bar provides quick access to File, View, Users, Run, Tools, Window, and Help menu
commands.

3. Toolbar commands provide quick access to menu options.

4. The left pane displays the instruments connected to the CFX Maestro computer and provides
buttons from which you can operate the lid and view the status of the instruments.

5. The main pane displays the working window. The default working window on the Home screen
is the Startup Wizard.

6. The status bar displays names of the connected instruments and the logged in user.

16 | CFX Maestro Software

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File Menu Commands

New — opens a dialog box from which you can choose to create a new protocol, plate, or gene study.

Open — opens a dialog box from which you can choose to navigate to and open an existing
protocol, plate, data file, gene study, LIMS file, run from a stand-alone instrument (stand-alone run),
or PrimePCR™ run file.

Recent Data Files — displays a list of recently opened PCR files.

Repeat a Run — opens Windows Explorer to the location of saved PCR files, in which you can
locate a run to repeat.

Exit — closes CFX Maestro.

View Menu Commands

Application Log — displays a software usage log from initial installation to the current day.

Run Reports — displays a list of run reports.

Startup Wizard — displays the Startup Wizard in the main pane.

Run Setup — displays the Run Setup window in the main pane.

Instrument Summary — displays the Instrument Summary window in the main pane.

Detected Instruments — toggles between displaying and not displaying connected instruments in
the left pane. By default, the software displays connected instruments in the left pane.

Toolbar — toggles between displaying and not displaying the toolbar on the top of the screen. By
default, the software displays the toolbar.

Status Bar — toggles between displaying and not displaying the status bar on the bottom of the
screen. By default, the software displays the status bar.

Show — opens a dialog box from which you can

n View or block the Status log.

n Open and view the CFX Maestro data folder.

n Open and view the user’s data folder.

n Open and view the LIMS file folder.

n Open and view the PrimePCR folder.

n View the run history.

n View the properties of all connected instruments.

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Chapter 4 The Home Window

User Menu Commands

Select User — opens the Login screen on which you can select a user from the User Name
dropdown list and log in to the application.

Change Password — opens the Change Password dialog box, in which users can change their
CFX Maestro software password.

User Preferences — opens the User Preferences dialog box, in which users can change the
default settings for

n Sending and receiving email notification upon run completion

n Saving data files

n Creating protocols via the Protocol Editor or the Protocol AutoWriter

n Creating plates

n Analyzing data

n Performing gene expression analysis

n Determining the quality of the data

n Exporting CFX instrument data

User Administration — opens the User Administration dialog box, in which administrators can
create users, modify role permissions, and assign roles to users.

Bio-Rad Service Login — for Bio-Rad technical service staff use only. Do not select this command.

Run Menu Commands

User-defined Run — opens the Run Setup window, in which you can set up a user-defined
protocol and plate, and then run a PCR experiment on selected instruments.

PrimePCR Run — opens the Start Run tab in the Run Setup window with the default PrimePCR
protocol and plate layout loaded based on the selected instrument.

End-Point Only Run — opens the Start Run tab in the Run Setup window with the default end-
point protocol and plate layout loaded based on the selected instrument.

Qualification Run — opens the Start Run tab in the Run Setup window with the default Bio-Rad
qualification protocol and plate layout loaded for the selected instrument.

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Tools Menu Commands

Master Mix Calculator — opens the Master Mix Calculator, in which you can create a reaction
mixture and print the calculations.

Protocol AutoWriter — opens the Protocol AutoWriter dialog box, in which you can easily create a
new protocol.

Ta Calculator — opens the Ta Calculator, in which you can easily calculate the annealing
temperature of primers.

Dye Calibration Wizard — opens the Dye Calibration wizard, in which you can calibrate an
instrument for a new fluorophore.

Reinstall Instrument Drivers — reinstalls the drivers that control communication with Bio-Rad’s
real-time PCR systems.

Zip Data and Log Files — opens a dialog box in which you can select files to condense and save
in a zipped file for storage or to email.

Batch Analysis — opens the Batch Analysis dialog box, in which you can set parameters for
analyzing more than one data file at a time.

Options — opens a dialog box in which you can

n Configure your email server settings.

n Configure export settings for LIMS and other data files.

Help Menu Commands

Tip: The Help menu is available on the menu bar in all CFX Maestro software windows.

Contents — displays the Contents tab in the CFX Maestro Help system.

Index — displays the Contents tab in the CFX Maestro Help system.

Search — displays the Contents tab in the CFX Maestro Help system.

Open User Guide — opens a PDF of this guide.

Additional Documentation — provides access to other CFX Maestro PDF documents and CFX
instrument guides.

Release Notes — opens the Release Notes document for the installed CFX Maestro software
version.

Video Resources — opens a web site where video resources, such as instructional videos, reside.

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Chapter 4 The Home Window

qPCR Applications and Technologies Web Site — opens Bio-Rad’s qPCR Applications &
Technologies web site, from which you can learn more about real-time PCR (qPCR).

qPCR Reagents Web Site — opens Bio-Rad’s PCR and qPCR reagents web site, from which you
can order PCR reagents, supermixes, dyes, and kits.

qPCR Plastic Consumables Web Site — opens Bio-Rad’s PCR Plastics and Consumables web
site, from which you can order PCR plates, plate seals, tubes and caps, and other plastics
accessories.

Software Web Site — opens Bio-Rad’s PCR Analysis Software web site, from which you can order
updated versions of Bio-Rad’s CFX Maestro software.

Check For Updates — opens the Software and Instrument Updates dialog box. When the CFX
Maestro computer is connected to the internet, the software detects whether new software or
instrument updates are available and can automatically perform the necessary updates.

About — displays CFX Maestro copyright and version information.

Toolbar Commands

— opens Windows Explorer, in which you can navigate to and open a data file or a gene
study file.

— opens the Master Mix Calculator.

— opens the Run Setup window.

— opens the Run Setup window with the default PrimePCR protocol and plate layout loaded
based on the selected instrument.

— opens the Startup Wizard.

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The Startup Wizard

When CFX Maestro starts, the working pane displays the Startup Wizard. From the Startup Wizard
you can

n Select an instrument from the detected instruments and set up a user-defined or PrimePCR
run.

n Open and repeat a run.

n Open a data file to analyze results from a single run or a gene study file for results from
multiple gene expression runs.

These tasks are explained in detail in the chapters that follow.

Status Bar
The left side of the status bar at the bottom of the main software window displays the current status
of the detected instruments. The right side of the status bar displays name of the current user and
the date and time.

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Chapter 4 The Home Window

Detected Instruments Pane

The Detected Instruments pane displays each instrument that is connected to the CFX Maestro
computer. By default, each instrument appears as an icon, and its serial number appears as its
name. The list of instruments also displays individual blocks (Block A and Block B) for each
dual-block reaction module installed on a C1000 Touch™ or S1000™ thermal cycler.

A red alert appears on instruments that require a system update. Until the system is updated, you
cannot use the instrument. For more information about system updates, see Performing Software and
Instrument Updates on page 28.

For example, the following image displays five detected instruments:

n One C1000™ Touch thermal cycler with a CFX96™ reaction module (SIM42465)

n One CFX Connect™(SIM81435) thermal cycler

n One S1000 thermal cycler with a dual 48-well block (SIM58851.A and SIM58851.B), which is
connected to the C1000 Touch thermal cycler (S83878)

n One C1000 Touch thermal cycler (SIM84046) with a CFX96 reaction module

Tip: This instrument requires an update. Until the system is updated, users cannot use this
instrument.

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From this pane you can do the following:

n View the properties and calibrated dyes for a selected instrument.

For information about instrument properties, see Viewing the Properties of an Instrument on
page 26.

n View the status of a connected instrument.

n Open the motorized lid on the selected instrument.

n Close the motorized lid on the selected instrument.

n View the status of all connected instruments.

To view the status of a connected instrument

u In the Detected Instruments pane, select target instrument and do one of the following:

n Click View Status in the Selected Instrument section.

n Right-click and select View Status on the menu that appears.

The Run Details dialog box appears displaying the Run Status tab. The status of the selected
instrument appears below the run status pane, for example:

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Chapter 4 The Home Window

To open or close the lid of an instrument

u In the Detected Instruments pane, select target instrument and do one of the following:

n Click Open Lid or Close Lid in the Selected Instrument section.

n Right-click and select the appropriate action on the menu that appears.

n Open the Run Details dialog box, select Run Status tab, and click Open Lid or Close Lid.

To view the status of all detected instruments

u Do one of the following:

n In the All Instruments section in the Detected Instruments pane, click View Summary.

n On the menu bar, select View > Instrument Summary.

The Instrument Summary dialog box appears:

Tip: If the system detects only one connected instrument, the All Instruments section does
not appear in the Detected Instruments pane. To view the instrument summary for a single
instrument, select View > Instrument Summary.

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Instrument Summary Toolbar Controls

Table 5 lists the controls and functions in the Instrument Summary toolbar.

Table 5. Instrument Summary Toolbar Controls

Button Button Name Function

Create a new Run Creates a run on the selected block by
opening the Run Setup window.

Stop Stops the current run on selected blocks.

Pause Pauses the current run on selected blocks.

Resume Resumes the run on selected blocks.

Flash Block Indicator Flashes the indicator LED on the lid of the
selected blocks.

Open Lid Opens the selected block’s motorized lid.

Close Lid Closes the selected block’s motorized lid.

Hide Selected Blocks Hide the selected blocks in the Instrument

Summary list

Show All Blocks Show the selected blocks in the Instrument

Summary list

Show Select which blocks to show in the list.

Select one of the options to show all
detected blocks, all idle blocks, all blocks
that are running with the current user, or all
running blocks

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Chapter 4 The Home Window

Viewing the Properties of an Instrument

From the Detected Instruments pane you can view details about a selected instrument, including its
properties, the status of its shipping screw, and a list of its calibrated dyes (fluorophores).

To view instrument properties

u In the Detected Instruments pane, right-click the target instrument and select Properties on the
menu that appears.

Properties Tab

The Properties tab lists technical details about the selected instrument including the model, serial
numbers of its components, and firmware versions. The default name of the instrument (its serial
number) appears in many locations, including the Detected Instruments pane and in the header
bar of the Instrument Properties dialog box. You can rename the instrument in order to more easily
identify it.

To rename an instrument

u In the Instrument Properties tab, type a name in the Rename box at the top of the Properties
tab and click Rename.

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The new name appears in the Nickname row in the Properties tab as well as in the Instrument
Properties header bar and the Detected Instruments pane.

Shipping Screw Tab

The Shipping Screw tab displays the current status of the shipping screw for the selected
instrument (Removed or Installed). The tab also includes instructions for installing or removing the
red shipping screw.

Tip: If the software detects the shipping screw, the Instrument Properties dialog box
automatically displays the Shipping Screw tab. Follow the instructions to remove the screw.

Note: You must remove the shipping screw before you can use the instrument. For more
information, see the Instrument Guide for the selected instrument.

Calibrated Dyes Tab

The Calibrated Dyes tab displays the calibrated fluorophores and plates for the selected
instrument.

To see detailed information about a calibration, click its Info button in the Detail column.

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Chapter 4 The Home Window

Before You Begin

Performing Software and Instrument Updates

When connected to the internet, CFX Maestro software automatically checks for updates each time
it starts. The software also determines whether updates are available for any connected
instrument. If any update is detected, CFX Maestro can automatically perform the update.

When updates are available, the Software and Instruments Updates dialog box automatically
appears.

Important: Systems requiring an update appear with a red alert. You cannot use the system
until you perform the requisite update.

When both software and system updates are performed, the software is updated first. In order to
download the latest updates, the computer running CFX Maestro software must be connected to
the internet. Close all data analysis windows and ensure that all instruments are idle before
beginning the update.

When the software update completes, CFX Maestro restarts and then begins the instrument
updates. CFX Maestro determines whether the instrument software is compatible with the version
of CFX Maestro software that is installed. If the instrument software is not compatible, this update
begins automatically. No internet connection is required for this operation.

Note: You must restart the instrument after the update completes.

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To perform software and system updates

u In the Software and Instrument Updates dialog box, do one of the following:

n If either a software or system update is available, select the appropriate checkbox and
click Update.

n If both software and instrument updates are available, select both checkboxes and click
Update.

Tip: In this case, updating the software requires an instrument update for any
connected instruments to ensure proper communication.

To hide the Software and Instrument Updates dialog box alert

u To hide the Updates dialog box when new updates are available, clear the Automatically
show this window when updates are available checkbox.

Important: If an attached instrument requires an update, the Software and Instrument

Updates dialog box automatically appears even if this checkbox is cleared. You must
perform the requisite update in order to run experiments on that instrument.

To display the Updates dialog box, on the Home window select Help > Check for Updates and
select the Notify when updates are available checkbox.

Setting User Preferences

Tip: It is not required to perform these tasks in order to use CFX Maestro software. You can
safely skip this section or perform these tasks at any time.

In CFX Maestro you can customize your working environment. If your administrator created
software users, each user can customize his or her working environment. If your administrator did
not create users, preference changes apply to everyone who logs in to CFX Maestro. (For
information about creating CFX Maestro users, see Appendix B, Managing CFX Maestro Users
and Roles.)

For example, in the Users > User Preferences menu, you can do the following:

n Set up email notification of run completion.

n Change the default settings for

o The location in which to save files

o The run setup files

o The file naming prefix

n Set the default parameters to use when creating a new protocol and plate.

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Chapter 4 The Home Window

n Set the default data analysis and gene expression parameters.

n Customize the default quality control parameters.

n Customize data export data parameters.

In the Tools menu, you can do the following:

n Create a master mix.

n Calibrate dyes for a specific instrument.

Note: The master mix and dye calibration are available to anyone who logs in to CFX Maestro.

This section explains how to perform these tasks in detail.

Setting Up Email Notification

You can connect CFX Maestro to your outgoing email server to send email notification of run
completion to a list of users. You can also choose to attach a data file and an analysis report to the
list of users. To set up the connection between CFX Maestro and your SMTP server, see
Connecting CFX Maestro to an SMTP Server on page 31.

To set up email notifications

1. Select Users > User Preferences to open the User Preferences dialog box.

The User Preferences dialog appears displaying the Email tab.

Note: You are informed if the system detects that you have not set up a valid SMTP server
for CFX Maestro. Click Configure Outgoing Email to open the Options dialog box and

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 Before You Begin

configure the email SMTP server. For more information, see Connecting CFX Maestro to
an SMTP Server on page 31.

2. In the To text box, type the email address of each person who you plan to inform of run
completion. All recipients will receive email after the run completes.

Note: You must enter each email address on a separate line. Press Enter or Return after
each address.

3. (Optional) In the cc text box, type the email address of any recipient to whom you plan to send
a copy of each email notification.

4. (Optional) By default, all recipients receive a copy of the data file as an attachment. Clear this
checkbox if you do not want to attach a copy the data file.

5. (Optional) Select Attach Analysis Report to attach a PDF of the analysis report to the email.

6. Click OK to save the changes and close the User Preferences dialog box.

To edit a recipient’s email address

u Modify the email address as necessary and click OK.

To remove an email recipient

1. Select the email recipient and press the Delete key.

2. Click OK to save the changes and close the dialog box.

Important: Clicking Restore Defaults in the User Preferences dialog box resets all
preferences on all tabs to the original factory settings. Take care when clicking this button.

Connecting CFX Maestro to an SMTP Server

Important: Some commercial webmail service providers (such as Yahoo! and Gmail) have
increased email security. If you use these accounts, you must enable the setting Allow less
secure apps in their account settings to enable CFX Maestro to send email. See the security
information for your webmail service provider for more information.

You must establish a connection from CFX Maestro to your email server before the software can
send email notification.

To connect CFX Maestro to an email server

1. Do one of the following:

n Select User > User Preferences and click Configure Outgoing Email on the Email tab.

n Select Tools > Options.

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Chapter 4 The Home Window

The Options dialog box appears displaying the Email tab.

2. Provide the following information for your company:

n SMTP Server Name — the name of the outgoing email server at your company.

n Port — the port number of your SMTP server. This is usually 25.

n Use SSL — Secure Sockets Layer (SSL) option. Some SMTP servers require this setting.
If it is not required at your company, clear this checkbox.

n Use Default “From” Address — the name of the email server at your company. Some
SMTP servers require all sent email to have a “from” address that is from a certain
domain, for example, [email protected]. If that is the case, clear this checkbox
and provide a valid email address.

n Authentication Required — if your site requires account authentication, verify that this
checkbox is selected.

n User Name — the name of the authenticated account. This is required only if
Authentication Required is selected.

n Password — the password for the authenticated account. This is required only if
Authentication Required is selected.

3. To verify that the SMTP server settings are correct, enter a valid email address in the Test
Email Address text box and click Test Email.

Note: Some SMTP servers do not allow attachments and others allow attachments only
up to a specific size. If you plan to email data files and/or reports using CFX Maestro,
select Test Attachment and set Attachment Size in MB to 5 megabytes (MB) or more.

4. Click OK to save the changes and close the dialog box.

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 Before You Begin

Changing the Default File Settings

In the Files tab on the User Preference dialog box, you can change the following:

n The default location in which to save CFX Maestro files

n The default files for run setup

n The default file naming parameters

To change the default file settings

1. Select Users > User Preferences to open the User Preferences dialog box.

2. In the User Preferences dialog box, select the Files tab.

3. In the Default Folder for File Creation section, navigate to and select a default folder in which
you want to save new files. You can select a different location for each file type:

n Protocol

n Plate

n Data File

n Gene Study

4. In the File Selection for Run Setup section, navigate to and select the target protocol and plate
files to appear when you open the Experiment Setup window.

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Chapter 4 The Home Window

5. In the Data File section, define the prefix and/or suffix for data files. For any part, select a new
value from its dropdown list. You can also provide custom prefix and suffix values in the Prefix
and Suffix text boxes.

CFX Maestro displays a preview of the file name below the selection boxes.

6. Click OK to save the changes and close the dialog box.

Important: Clicking Restore Defaults in the User Preferences dialog box resets all
preferences on all tabs to the original factory settings. Take care when clicking this button.

Setting the Default Protocol Parameters

To set default protocol parameters for the Protocol Editor and Protocol AutoWriter

1. Select Users > User Preferences to open the User Preferences dialog box.

2. In the User Preferences dialog box, select the Protocol tab.

3. In the Protocol Editor section, specify values for the following settings that appear in the
Protocol Editor:

n Sample volume — the volume of each sample in the wells (in µl).

n Lid Shutoff temperature — the temperature in ºC at which the lid heater turns off during a
run.

4. In the Protocol AutoWriter section, specify values for the following settings that appear in the
Protocol AutoWriter:

n Annealing temperature — the temperature in ºC for experiments that use iProof™ DNA
polymerase, iTaq™ DNA polymerase, or other polymerases.

34 | CFX Maestro Software

 Before You Begin

n Amplicon length — the length of the amplicon in bp.

5. Click OK to save the changes and close the dialog box.

Important: Clicking Restore Defaults in the User Preferences dialog box resets all
preferences on all tabs to the original factory settings. Take care when clicking this button.

Setting Default Plate Parameters

Changes that you make to the Plate tab are available to all users of the software. Changes that you
make during plate setup are available to users after you save and close the plate file.

In the User Preferences dialog box you can do the following:

n Set default plate parameters.

n Add new target, sample, and biological group names to their respective libraries.

n Delete target, sample, and biological group names from their respective libraries.

To set the default plate parameters

1. Select Users > User Preferences to open the User Preferences dialog box.

2. In the User Preferences dialog box, select the Plate tab.

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Chapter 4 The Home Window

3. Specify values for the followings settings for a new plate file. These values appear in the Plate
Editor window:

n Plate type

n Plate size

n Units — the concentration of the starting template for wells that contain standards.

CFX Maestro uses these units to create a standard curve in the Data Analysis
Quantification tab.

n Scientific notation — when selected, CFX Maestro displays the concentration units in
scientific notation.

n Scan mode — the number or type of channels to scan during a run.

n Fluorophores — the default fluorophores that appear in the Plate Editor well loading
controls.

n Libraries — the targets, samples, and biological groups that you typically use in your
experiments:

o Target names — the names of target genes and sequences.

o Sample names — the names of experiment samples or an identifying characteristic

for the samples (for example, Mouse1, Mouse2, Mouse3).

o Biological group names — the names for groups of similar samples that have the
same treatment status or conditions (for example, 0Hr, 1Hr, 2Hr).

4. Click OK to save the changes and close the dialog box.

To add a new target, sample, or biological group name

u In the appropriate library box, type the name for the target, sample, or biological group and
click OK.

To delete a target, sample, or biological group name

u In the appropriate library box, select the name and press the Delete key and then click OK.

Important: Names that you remove from the library are removed from the software and are no
longer available to users. To restore the default CFX Maestro names, click Restore Defaults.
Clicking Restore Defaults in the User Preferences dialog box resets all preferences on all tabs
to the original factory settings. Take care when deleting default CFX Maestro names and when
clicking this button.

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Setting Default Data Analysis Parameters

To set default Data Analysis parameters

1. Select Users > User Preferences to open the User Preferences dialog box.

2. In the User Preferences dialog box, select the Data Analysis tab.

3. In the Analysis Mode section, select the mode in which to analyze the data (either Fluorophore
or Target).

4. In the PCR Quantitation section, set default parameters for the following options:

n Baseline Setting — the baseline method for analysis mode.

n Cq Determination Mode — the mode in which Cq values are calculated for each
fluorescence trace (either regression or single threshold).

n Threshold Calc. Mode — the end-point target amount.

The default is Auto. That is, the software automatically calculates the end-point target. To
set a specific threshold, clear the Auto checkbox and enter your end-point amount,
calculated in relative fluorescence units (or RFU). The maximum value is 65000.00 RFUs.
Data files for subsequent runs will use this threshold setting.

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Chapter 4 The Home Window

n Baseline Calc. Mode — the baseline value for all traces.

The default is Auto. That is, the software automatically calculates the baseline for all
traces. To set a specific baseline value, clear the Auto checkbox and enter minimum and
maximum values for the cycle range (1 to 9999). Data files for subsequent runs will use
this cycle range.

n Log View — determines how the software displays the amplification data:

o On — the amplification data are displayed in a semilogarithmic graph.

o Off — (the default) the amplification data are displayed in a linear graph.

5. In the End Point section, select the number of end cycles to average when calculating the end-
point calculations:

n PCR run — the number of end cycles to average for quantification data (default is 5).

n End Point Only run — the number of end cycles to average for end-point data (default is
2).

6. In the Melt Curve section, select the peak type to detect (either positive or negative).

7. In the Well Selector section, select how to display well labels (by sample type, target name, or
sample name).

8. Click OK to save the changes and close the dialog box.

Important: Clicking Restore Defaults in the User Preferences dialog box resets all
preferences on all tabs to the original factory settings. Take care when clicking this button.

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 Before You Begin

Setting Default Gene Expression Data File Parameters

To set the default parameters for a new gene expression data file

1. Select Users > User Preferences to open the User Preferences dialog box.

2. In the User Preferences dialog box, select the Gene Expression tab.

3. Specify the values for the following settings:

n Relative to — graphs the gene expression data relative either to a control (originating at
1) or to zero:

o Zero — the software ignores the control. This is the default when no control sample is
assigned in the Experiment Settings window.

o Control — the software calculates the data relative to the control sample assigned in
the Experiment Setup window.

n X-axis — graphs the sample or the target on the x-axis.

n Y-axis — graphs linear, log2, or log10 scale on the y-axis.

n Scaling — the scaling option for the graph (the default option is unscaled):

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Chapter 4 The Home Window

o Highest — the software scales the graph to the highest data point.

o Lowest — the software scales the graph to the lowest data point.

o Unscaled — the software presents the data unscaled in the graph.

n Mode — the analysis mode, either relative quantity (∆Cq ) or normalized expression
(∆∆Cq ).

n Error Bar — the data variability presented as either the standard deviation (Std. Dev.) or
the standard error of the mean (Std. Error Mean).

n Error Bar Multiplier — the standard deviation multiplier used to graph the error bars
(default is 1).

You can increase the multiplier to either 2 or 3.

n Sample Types to Exclude — the sample types to exclude from the analysis.

You can select one or more sample to exclude from the analysis. To exclude all sample
types, clear the checkboxes of any selected sample types.

4. Click OK to save the changes and close the dialog box.

Important: Clicking Restore Defaults in the User Preferences dialog box resets all
preferences on all tabs to the original factory settings. Take care when clicking this button.

Customizing Quality Control Rules

In CFX Maestro, you can set quality control rules, which are applied to data in the Data Analysis
window. The software validates the data against the rules that you set.

Note: By default, all quality control rules are enabled.

Tip: You can easily exclude wells that fail a QC parameter from analysis in the QC module of
the Data Analysis window.

To customize quality control rules

1. Select Users > User Preferences to open the User Preferences dialog box.

2. In the User Preferences dialog box, select the QC tab.

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 Before You Begin

where:

n NTC — no template control

n NRT — no reverse transcriptase control

n Efficiency — reaction efficiency

n Std Curve R^2 — R square value for the standard curve

n Replicate group Cq Std Dev — standard deviation calculated for each replicate group

3. For each QC rule, do one of the following:

n To use its default value, do nothing.

n To change its value, click its Value text box, type a new value, and press the Enter key.

n To disable the rule, clear its Use checkbox.

4. Click OK to save the changes and close the dialog box.

Important: Clicking Restore Defaults in the User Preferences dialog box resets all
preferences on all tabs to the original factory settings. Take care when clicking this button.

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Chapter 4 The Home Window

Customizing Data Export Parameters

You can export CFX Maestro data in the following formats:

n Text (.txt)

n CSV (.csv)

n Excel 2007 (.xlsx)

n Excel 2003 (.xls)

n XML (.xml)

n HTML (.html)

You can specify the type of data to export and customize the output of the exported data.

To customize data export parameters

1. Select Users > User Preferences to open the User Preferences dialog box.

2. In the User Preferences dialog box, select the Custom Export tab.

3. On the Export Format dropdown list, select a format in which to export the data.

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 Before You Begin

4. In the Data to Export section, select or clear the checkboxes for the type of data to export. The
selected items appear in the Exported Columns list box.

Note: By default, the run information is included in the header. Clear this checkbox if you
do not want the run information included.

5. You can change the output display order of the selected items.

In the Exported Columns list box, highlight the item and then click the arrow buttons to the left
of the list to move it up or down.

6. Optionally, you can change the output column names of the selected items:

a. Click Customize Column Names.

The Column Name Customizer dialog box appears.

b. For each default column name that you want to change, type the new name in its Custom
Name field.

c. Do one of the following:

n Click OK to save the changes and return to the Custom Export tab. The new name
appears in parentheses beside the default column name in the Exported Columns list
box.

n Click Cancel to clear the changes and return to the Custom Export tab.

7. Click OK to save the changes and close the dialog box.

Important: Clicking Restore Defaults in the User Preferences dialog box resets all
preferences on all tabs to the original factory settings. Take care when clicking this button.

Creating a Reaction Master Mix

Using CFX Maestro’s Master Mix Calculator, you can easily calculate the required volume of each
component in your master mix. You can print the master mix calculation table to your default
printer, and save the calculations for each target for later use.

To create a reaction master mix using the Master Mix Calculator

1. To open the Master Mix Calculator, do one of the following:

n Select Tools > Master Mix Calculator.

n Click Master Mix Calculator on the toolbar.

The Master Mix Calculator appears.

User Guide | 43
Chapter 4 The Home Window

2. In the Reaction section, select a detection method:

n SYBR® Green/EvaGreen

n Probes

3. To create a new target, in the Target section click Create New. A new target name appears in
the target dropdown list.

4. (Optional) To change the default target name:

a. Highlight the target’s name in the dropdown target list.

b. Type a new target name in the Target box.

c. Press the Enter key.

5. Adjust the starting and final concentrations for the forward and reverse primers and any
probes.

44 | CFX Maestro Software

 Before You Begin

6. In the Master Mix Setup section, adjust the values for

n Number of reactions to run

n Reaction volume per well

n Template volume per well

n Supermix concentration per well

n Excess reaction volume per well

7. (Optional) Perform steps 2–6 for as many targets as necessary.

8. In the Choose Target to Calculate section, select the target to calculate.

Tip: You can calculate only one or several or all targets at the same time.

The calculated volumes of the required components for each selected target appear in the
master mix table.

9. Click Set as Default to set the quantities input in the Target and Master Mix Setup sections as
new defaults.

10. Click OK to save the contents of the Master Mix Calculator dialog box.

To print the master mix calculations table

u To print a master mix calculations table, click Print.

The calculations table prints to your default printer.

To save the master mix calculations table as a PDF

u Change your default printer to a PDF driver and click Print on the Master Mix Calculator.

To delete targets

u Select the target using the dropdown target list and click Remove.

Important: Removing a target from the target list also removes it from any master mix
calculations it is used in. Take care when deleting a target.

User Guide | 45
Chapter 4 The Home Window

Calibrating New Dyes

The CFX96 Touch™, CFX96 Touch Deep Well, and CFX Connect™ systems are factory calibrated
for commonly used fluorophores in white-well and clear-well plates. The CFX384 Touch™ system
is factory calibrated for the same fluorophores in white-well plates only. Table 6 lists the
fluorophores and channel for which each instrument is calibrated.

Note: The CFX96 Touch, CFX96 Touch Deep Well, CFX Connect, and CFX384 Touch
systems also include a channel dedicated to FRET chemistry. This channel does not require
calibration for specific dyes.

Table 6. Factory calibrated fluorophores, channels, and instruments

Excitation, Detection,
Fluorophores Channel nm nm Instrument
FAM, SYBR® Green I 1 450–490 515–530 CFX96 Touch, CFX96
Touch Deep Well, CFX
Connect, and CFX384
Touch systems

VIC, HEX, CAL Fluor 2 515–535 560–580 CFX96 Touch, CFX96

Gold 540, Cal Fluor Touch Deep Well, CFX
Orange 560 Connect, and CFX384
Touch systems

ROX, Texas Red, 3 560–590 610–650 CFX96 Touch, CFX96

CAL Fluor Red 610, Touch Deep Well, and
TEX 615 CFX384 Touch systems

CY5, Quasar 670 4 620–650 675–690 CFX96 Touch, CFX96

Touch Deep Well, and
CFX384 Touch systems

Quasar 705, Cy5.5 5 672–684 705–730 CFX96 Touch and CFX96

Touch Deep Well systems
only

46 | CFX Maestro Software

 Before You Begin

To calibrate new dyes for CFX systems

1. In the Home window, select a target instrument in the Detected Instruments pane.

2. Select Tools > Calibration Wizard to open the Dye Calibration wizard.

Fluorophores already calibrated for the target instrument appear in the Calibrated
Fluorophores table.

3. In the Calibrate New or Existing Fluorophores section, select the fluorophore to calibrate from
the dropdown list.

If the fluorophore name is not included in the list, type its name in the text box to add it to the
list.

4. Select the plate type for the fluorophore.

If the plate type is not included in the list, type the name in the text box to add it to the list.

5. Select a channel for the fluorophore.

6. Select a plate column for the fluorophore.

7. (Optional) Type a color to associate with the fluorophore.

8. Click Add to Plate to add the fluorophore.

9. (Optional) Repeat steps 3—8 to add each fluorophore you plan to calibrate for the plate.

User Guide | 47
Chapter 4 The Home Window

10. When you finish adding fluorophores, click View Plate to open the Pure Dye Plate Display
window.

Use this window as a guide for loading dyes into the plate.

11. Prepare a 96- or 384-well plate for dye calibration:

a. Pipette dye solution into each well, following the pattern shown in the Pure Dye Plate
Display.

b. For each fluorophore, fill four wells with 50 µl (96-well plate) or 30 µl (384-well plate) of
300 nM dye solution. Notice that at least half the plate contains blank wells.

c. Seal the plate using the sealing method you will use in your experiment.

12. Place the calibration plate in the block and close the lid.

13. In the Dye Calibration wizard, click Calibrate and then OK to confirm that the plate is in the
block.

14. When CFX Maestro software completes the calibration run, a dialog box appears. Click Yes to
finish calibration and open the Dye Calibration Viewer.

15. Click OK to close the window.

48 | CFX Maestro Software

Chapter 5 Creating Protocols
A protocol is set of steps that are executed in a specific sequence. In CFX Maestro™ software, all
of the steps are associated with options on the instrument. For example, the steps instruct the
instrument to control block and lid temperature, apply a temperature difference across the block,
take a plate read, or perform a melt curve analysis. Each option is specified for different plate and
run types.

CFX Maestro provides two options for creating protocols: Protocol Editor and Protocol AutoWriter.

The Protocol Editor features include the following:

n Standard protocol controls to quickly create protocols

n Ability to quickly calculate a gradient for the selected number of rows

n Ability to quickly calculate run time for the selected plate type

n Ability to edit protocol steps

n Ability to save protocols for reuse

n Ability to print the protocol to a default printer

The Protocol AutoWriter automatically generates a customized PCR protocol with hot start, initial
denaturation, annealing, and extension steps using parameters that you provide. You can then
view a graphical representation of the suggested protocol and edit, run, or save the protocol.

User Guide | 49
Chapter 5 Creating Protocols

Protocol Editor Window

Use the Protocol Editor to create, open, review, and edit a protocol. By default, the Protocol Editor
opens displaying a generic real-time 2-step protocol for a 96-well plate.

LEGEND

1. The menu bar provides quick access to the File, Settings, and Tools menu commands.

2. The toolbar provides quick access to save and print the protocol, determine where to insert a
step, set sample volume, and view estimated protocol run time.

3. The main pane displays a graphical representation of the protocol.

4. The lower pane displays the protocol outline.

5. The left pane displays the protocol controls that you can add to customize the protocol.

File Menu Commands

Save — saves the current protocol.

Save As — saves the current protocol with a new name or in a new location.

Close — closes the Protocol Editor.

50 | CFX Maestro Software

 Protocol Editor Window

Settings Menu Command

Lid Settings — opens the Lid Setting dialog box in which you can change or set the lid
temperature.

Tools Menu Commands

Gradient Calculator — opens a dialog box from which you can select the block type for a gradient
step. The default is 96 wells.

Run time Calculator — opens a dialog box from which you can select the plate type and scan
mode in order to calculate the estimated run time in the Run Setup window. The default is 96 wells,
all channels.

Toolbar Commands

— saves the current protocol file.

— prints the selected window.

— use this command to select where to insert steps relative to the currently
selected step.

— use this command to enter a sample volume in µl. Sample volumes

differ depending on the type of block:

n For a 96–deep well block the range is 0–125 µl.

n For a 96–well block the range is 0–50 µl.

n For a 384–well block the range is 0–30 µl.

See Section XXXX for more information about sample volume.

— displays the estimated run time based on the protocol steps, ramp rate,
and the type of block selected.

— displays Help information about protocols.

User Guide | 51
Chapter 5 Creating Protocols

Protocol Editing Controls

The left pane of the Protocol Editor window comprises controls that you can use to create
protocols.

Each control consists of a set of parameters that represent a step in the protocol. You can modify
each parameter and add or remove them to customize your protocol. This section describes the
options in each control.

n Insert Step — inserts a step before or after the selected step. You
can edit the temperature and hold time values either in the
protocol’s graphical display or in the protocol outline.

n Insert Gradient — inserts a gradient step based on the type of well

block selected in the gradient calculator. You can edit the gradient
range in the Gradient pane that appears when a gradient step is
inserted.

n Insert GOTO — inserts a cycling (loop) step, which informs the

software to repeat specific steps in sequence for a specified
number of cycles. The repetitions begin after the first cycle is
complete. For example, you can inform the software to perform 39
repetitions of steps 2–4. After the final repetition, the software will have performed steps 2–4 a
total of 40 times. You can edit the return-to (GOTO) step and the number of cycles either in the
graphical display or in the protocol outline.

n Insert Melt Curve — inserts a melt curve read step.

n Insert Plate Read to Step — adds a plate read command to the selected step. A plate read
measures the amount of fluorescence at the end of a cycle. The plate read step is generally
the last step in a GOTO loop.

Tip: After you add a plate read command to a step, the button changes to Remove Plate
Read when you select the step.

n Remove Plate Read — removes a plate read command from the selected step.

Tip: After you remove a plate read command from a step, the button changes to Add Plate
Read to Step when you select the step.

n Step Options — opens the Step Options dialog box and displays the options available for the
selected step. See Step Options on page 53 for detailed information about the step options.

Tip: You can also access the Step Options by right-clicking the step in the graphic display.

n Delete Step — deletes the selected step from the protocol.

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 Protocol Editor Window

Step Options

Open the Step Options dialog box to view the options you can add, change, or remove from a step.

n Plate Read — when selected, adds a plate read to the step.

n Temperature — sets the target temperature for the selected step.

n Gradient — sets the gradient range for the step; the range is 1–24°C.

Note: A gradient runs with the lowest temperature in the front of the block (in this image,
row H) and the highest temperature in the back of the block (in this image, row A).

n Increment — the amount to increase (or decrease) the temperature of the selected step; this
value amount is added to the target temperature with each cycle. The range is ±0.1–10°C.

Note: To decrease the temperature, type a minus sign (–) before the numerical value
(for example, –5°C).

n Ramp Rate — the ramp rate for the selected step; the range depends on the block size.

n Time — the hold time for the selected step.

n Extend — the amount of time (in sec) to extend or decrease the selected step; this option is
added to the hold time in each cycle; the range is 1–60 sec.

n Beep — when selected, a beep sounds at the end of the step.

Tip: When you enter a number that is outside the option range, the software changes the
number to the closest entry within the range.

User Guide | 53
Chapter 5 Creating Protocols

Creating a Protocol in the Protocol Editor

Using the Protocol Editor, you can create custom protocol files. You can also edit and save
previously saved protocol files or sample protocol files shipped with CFX Maestro software.

To create a new protocol file, perform the following:

n Open a protocol file in the Protocol Editor.

Tip: You can open a new or existing protocol in the Protocol Editor.

n Set up the new protocol.

n Add steps to the protocol from the protocol controls pane.

n Edit the properties of the steps.

n Save the protocol.

Tip: To create a new protocol from a previously saved or sample protocol file, see Opening an
Existing Protocol in the Protocol Editor on page 55.

Opening a New Protocol File in the Protocol Editor

CFX Maestro offers multiple options to open a new protocol file:

n From the Home window

n From the Startup Wizard dialog box

n From the Run Setup dialog box

To open a new protocol file from the Home window

u Select File > New > Protocol.

The Protocol Editor window opens, displaying the default protocol file.

Tip: For information about setting your default protocol, see Changing the Default File
Settings on page 33.

To open a new protocol file from the Startup Wizard

1. In the Home window, do one of the following to open the Startup Wizard if it is not in view:

n Select View > Startup Wizard.

n Click Startup Wizard on the toolbar.

By default, the Startup Wizard displays the Run setup tab with the CFX96™ instrument type
selected.

54 | CFX Maestro Software

 Creating a Protocol in the Protocol Editor

2. If necessary, select the instrument type from the dropdown list.

3. Click User-defined as the run type.

The Run Setup dialog box opens to the Protocol tab and displays the default protocol file.

4. Click Create New.

The Protocol Editor window opens, displaying the default real-time protocol.

To open a new protocol from the Run Setup dialog box

1. In the Home window, do one of the following to open the Run Setup dialog box:

n Select Run > User-defined Run.

n Click User-defined Run Setup on the toolbar.

The Run Setup dialog box opens to the Protocol tab and displays your default protocol file.

2. Click Create New.

The Protocol Editor window opens, displaying the default real-time protocol.

Opening an Existing Protocol in the Protocol Editor

CFX Maestro provides sample protocol files that you can edit and save as custom new protocols.
You can also create a new protocol from an existing custom protocol.

To open a sample protocol file

1. In the Home window, select File > Open > Protocol.

By default, Windows Explorer opens to the location of the CFX Maestro Sample files folder.

2. Open the Sample files folder. You see the following folders:

n ConventionalProtocols — contains example protocol files for traditional PCR analysis.

n DataFiles — contains example data files that you can use to explore CFX Maestro’s
features.

n MeltCalibration — contains example protocol files for use with Bio-Rad’s Precision Melt
Analysis software.

n Plates — contains example plate files.

n RealTimeProtocols – contains example protocol files for real-time PCR analysis.

3. Open the protocol folder for the type of run you plan to perform, either ConventionalProtocols
or RealTimeProtocols.

User Guide | 55
Chapter 5 Creating Protocols

4. Select the protocol of choice and click Open.

The sample protocol opens in the Protocol Editor window.

5. Select File > Save As and save the protocol with a new name or in a new folder.

To open an existing protocol

1. In the Home window, do one of the following:

n Select File > Open > Protocol, navigate to and select the target protocol, and click Open.

n Open the Startup Wizard and do one of the following:

o To edit the displayed protocol, click Edit Selected.

o To edit another existing protocol, click Select Existing and navigate to the target file.

The protocol opens in the Protocol Editor window.

2. Select File > Save As and save the protocol with a new name or in a new folder.

Setting Up a New Protocol

Tip: If your protocol file includes the required parameters (for example, if you are editing an
existing plate file) you can skip this section. Proceed to Adding Steps to a Protocol on page 58.

New protocol files require the following parameters:

n Block type

n Scan mode for the chosen block type

n Lid temperature

n Sample volume

Setting the Block Type

CFX Maestro automatically calculates temperature increments for gradient steps based on the
block type.

Note: The plate type set in the Protocol Editor must be the same as the plate in the reaction
module.

To set the block type

u In the Protocol Editor window, select Tools > Gradient Calculator and choose the appropriate
plate type in the dropdown list that appears.

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 Creating a Protocol in the Protocol Editor

Selecting the Scan Mode for the Chosen Block Type

To determine the run time for the protocol, select the target block type and scan mode.

To select the block type and scan mode

u In the Protocol Editor window, select Tools > Run time Calculator and choose the appropriate
plate type and scan mode in the dropdown list that appears.

Adjusting the Lid Temperature

CFX Maestro sets the default lid temperatures as follows:

n 96–well instruments — 105.0°C

n 384–well instruments — 95.0°C

n 48–well instruments — 100.0°C

You can change the default settings or turn off the lid heater as necessary for the protocol.

Tip: You can change the default lid temperature in the User Preferences dialog box. See
Setting the Default Protocol Parameters on page 34.

To adjust the lid temperature

1. In the Plate Editor window, select Settings > Lid Settings.

The Lid Settings dialog box appears.

2. Do one of the following:

n Select User Defined and enter a temperature value in the text box.

n Select Turn Off Lid Heater.

3. Click OK to accept the changes and close the dialog box

Setting the Sample Volume

By default, CFX Maestro sets the sample volume for each well to 25 µl. Sample volumes differ
depending on the type of block, for example:

n 0–125 µl for a 96-deep well block

n 0–50 µl for a 96-well block

n 0–30 µl for a 384-well block

User Guide | 57
Chapter 5 Creating Protocols

The instrument uses one of two temperature control modes to determine when the sample reaches
the target temperature in a protocol:

n Calculated mode — when the sample volume is set to a volume appropriate for the block, the
instrument calculates the sample temperature based on the sample volume. This is the
standard mode.

n Block mode — when the sample volume is set to zero (0) µl, the instrument records the
sample temperature as the same as the measured block temperature.

To set the sample volume for a specific block

u In the Plate Editor window, type the correct value in the Sample Volume text box on the
toolbar.

Tip: You can change the default sample volume in the User Preferences dialog box. See
Changing the Default File Settings on page 33.

Adding Steps to a Protocol

To add a step to a protocol

1. Open the protocol in the Protocol Editor window.

2. Determine where to insert the new step. On the toolbar, select Before or After in the Step
dropdown list.

3. On the graph, select the step before or after which you plan to insert the new step.

4. In the left pane, click Insert Step.

5. To change the temperature or hold time, click the default value on the graph or the protocol
outline and type a new value.

6. (Optional) In the left pane, click Step Options to display the Step Options dialog box and
modify the available options for the selected step.

Tip: You can access the Step Options dialog box on the right-click menu in either the
graph pane or the protocol outline pane.

7. Click OK and then click Yes to save changes to the protocol.

The Save As dialog box appears

8. In the Save As dialog box, type a name for the new protocol file and click Save.

58 | CFX Maestro Software

 Creating a Protocol in the Protocol Editor

Inserting a Gradient Step

To insert a gradient step

1. Verify that the plate size for the gradient is the same as the block type of the instrument, 96-
well or 384-well.

2. If you have not yet done so, select the plate size for the gradient:

Select Tools > Gradient Calculator and choose the appropriate well type from the dropdown
list.

3. On the toolbar, select either Before or After from the Insert Step dropdown list.

4. In the graph or outline pane, select the step before or after which you plan to insert the
gradient step.

5. In the left pane, click Insert Gradient. The new gradient step is highlighted in the graph and the
outline pane, for example:

The temperature of each row in the gradient appears in the Gradient table in the right pane.

User Guide | 59
Chapter 5 Creating Protocols

6. To edit the gradient temperature range, do one of the following:

n Click the default temperature in the graph or outline pane and enter a new temperature.

n Click Step Options to enter the gradient range in the Step Options window.

n Change the Range value in the Gradient table.

7. To edit the hold time, click the default time in the graphic or text view and enter a new time.

8. Click OK and then Yes to save the changes.

Inserting a GOTO Step

Note: You cannot insert a GOTO step within a GOTO set; you cannot create nested GOTO
loops.

To insert a GOTO step

1. On the toolbar, select Before or After from the Insert Step dropdown list.

2. In the graph, select the step before or after which you plan to insert the GOTO step.

3. In the left pane, click Insert GOTO.

4. To edit the GOTO step number or number of GOTO repeats, select the default number in the
graph or outline pane and enter a new value.

5. Click OK and then Yes to save the changes.

Inserting a Melt Curve Step

Tip: You cannot insert a melt curve step inside a GOTO loop.

Note: The melt curve step includes a 30 sec hold at the beginning of the step that is not shown
in the protocol.

To insert a melt curve step

1. On the toolbar, select Before or After from the Insert Step dropdown list.

2. In the graph, select the step before or after which you plan to insert the melt curve step.

60 | CFX Maestro Software

 Creating a Protocol in the Protocol Editor

3. In the left pane, click Insert Melt Curve. The new melt curve step is highlighted in the graph
and the outline pane, for example:

4. To edit the melt temperature range or increment time, select the default number in the graph or
outline pane and enter a new value.

5. Click OK and then Yes to save the changes.

User Guide | 61
Chapter 5 Creating Protocols

Adding or Removing a Plate Read Step

Tip: After you add a plate read command to a step, the button changes to Remove Plate Read
when you select the step.

To add a plate read to a step

1. On the toolbar, select Before or After from the Insert Step dropdown list.

2. On the graph, select the step before or after which you plan to insert the plate read step.

3. In the left pane, click Add Plate Read to Step to add a plate read to the selected step.

4. Click OK and then Yes to save the changes.

To remove a plate read from a step

u On the graph, select the step that contains the plate read and click Remove Plate Read in the
left pane.

Changing Step Options

To change step options for a selected step

1. Select the target step in the graph or outline pane.

2. In the left pane, click Step Options to open the Step Options dialog box.

Alternatively, right-click the target step in either pane and select Step Options in the menu that
appears.

3. To add, modify, or remove options:

n Enter a value in the appropriate text box.

n Edit a value in the specific text box.

n Select or clear a checkbox.

4. Click OK to save the changes and close the Step Options dialog box.

5. Click OK and then Yes to save the protocol.

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 Creating a Protocol in the Protocol Editor

Deleting a Step
Important: You cannot undo this function. Take care when deleting steps.

To delete a step in the protocol

1. Select the step in the graph or outline pane.

2. In the left pane, click Delete Step to delete the selected step.

3. Click OK and then Yes to save the protocol.

Copying, Exporting, or Printing a Protocol

To copy a protocol

u Right-click the protocol outline and select Copy Protocol.

You can paste the outline into a .txt, .xls, .doc, or .ppt file.

To export a protocol

1. Right-click the protocol outline and select Export Protocol.

The Save As dialog box appears.

2. (Optional) In Windows Explorer, navigate to a folder in which to save the protocol file.

3. In File name, type a name for the exported protocol file.

4. Click Save.

To print a protocol

u Right-click the protocol outline and select Print.

You can print the protocol outline to your default printer.

User Guide | 63
Chapter 5 Creating Protocols

Creating a Protocol with the Protocol AutoWriter

Important: Bio-Rad does not guarantee that running a protocol created with the Protocol
AutoWriter will always result in a PCR product.

CFX Maestro’s Protocol AutoWriter automatically generates cycling protocols based on the
following input parameters:

n Amplicon length — the expected length of the PCR product

n Annealing temperature — the reaction Ta for the primers being used

If the Ta is unknown, you can use the Ta calculator to automatically calculate it based on your
primer sequences.

Note: The Ta is adjusted from the primer melting temperature (Tm) information that is
based on the selected enzyme and the protocol speed.

n Enzyme type — the DNA polymerase enzyme (iTaq™, iProof™ DNA polymerase, or Other)

If you use an enzyme other than iTaq or iProof DNA polymerase, you can enter additional
information, including the gradient range, hot-start activation time (in sec), and the final
extension time (in sec).

n Run speed — the reaction speed (standard, fast, or ultrafast)

The Protocol AutoWriter optimizes the protocol depending on the selected speed setting. The
total run time is determined by the number of steps and cycles, the incubation time at each
step, and the time it takes to reach uniformity at the target temperature.

Using parameters that you enter and standard PCR guidelines, the Protocol AutoWriter
automatically generates a customized PCR protocol with hot start, initial denaturation, annealing,
and extension steps. You can then view a graphical representation of the suggested protocol and
edit, run, or save the protocol.

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 Creating a Protocol with the Protocol AutoWriter

To create a new protocol using CFX Maestro’s Protocol AutoWriter

1. In the Home window, select Tools > Protocol AutoWriter.

The Protocol AutoWriter dialog box appears.

2. In the Enter Target Values/Enzyme section, do the following:

n Enter the annealing temperature (Ta ) for the primers, if known.

Tip: Using the Ta Calculator on page 66 for more information.

Note: For information about the calculations used in the Ta Calculator, see Breslauer
et al. 1986.

n Enter the amplicon length in base pairs (bp).

n Select an enzyme type from the list of options (iTaq™ DNA polymerase, iProof™ DNA
polymerase, or Other).

Tip: If you select Other as the enzyme type, the parameters in the Additional
Parameters (Optional) section become active.

User Guide | 65
Chapter 5 Creating Protocols

3. If you selected Other as the enzyme type, you can add any or all of the following parameters to
the protocol:

n Gradient range

n Hot start activation temperature

n Final extension time

4. In the Type section, move the sliding bar to select a protocol speed (Standard, Fast, or
Ultrafast). CFX Maestro adjusts the total run time.

5. Select the type of PCR to perform (Real-time PCR is the default).

With real-time PCR, CFX Maestro adds a plate read step to collect fluorescence data.

6. In the Preview section, review the protocol. You can make changes as needed.

7. Do one of the following:

n Click OK to save the new protocol. After saving it, the protocol opens in the Startup
Wizard. Click Edit Selected to make any changes to the protocol. For example, you might
need to change the lid temperature and sample volume.

n Click Cancel to close the window without saving the protocol.

Using the Ta Calculator

When the annealing temperature for the primer is unknown, you can use the Ta Calculator to
calculate the value. You can use the value in the Protocol AutoWriter or in the Protocol Editor to
create your protocol.

About the Ta Calculator

The Ta Calculator calculates the Tm value for each primer as well as the Ta value for the protocol at
standard speed.

The Ta for the protocol is based on the average primer Tm values with the following rules applied:

n If the difference between the primer Tm values is >4°C, the Ta = (lower of the two primer Tm
values + 2) – 4°C

n If the difference between the Tm values is ≤4°C, the Ta = (average of the primer Tm values) –
4°C

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 Using the Ta Calculator

Base Pair Counting Method

For each primer, the Ta Calculator uses the base pair counting method for sequences of 14 base
pairs (bp) or fewer.

Tm = ((w*A + x*T) * 2) + ((y*G + z*C) * 4)

where w, x, y, and z are the numbers of the bases A, T, G, and C in the sequence, respectively.

Nearest Neighbor Method

For sequences longer than 14 bp, the nearest neighbor method is used. In the nearest neighbor
method, the melting temperature calculations are based on the thermodynamic relationship
between entropy (order or a measure of the randomness of the oligonucleotide), enthalpy (heat
released or absorbed by the oligonucleotide), free energy, and temperature.

∆H = ∆G + T* ∆S

where:

n ∆H = Enthalpy value, Cal/Mole*K

n T = temperature, Kelvin

n ∆S = Entropy value, Cal/Mole*K

n ∆G = Gibbs free energy in Cal/Mole*K

The change in entropy and enthalpy is directly calculated by summing the values for nucleotide
pairs shown in Table 7 (Breslauer et al. 1986).

The relationship between the free energy and the concentration of reactants and products at
equilibrium is given by:

∆G = R*T*ln ((DNA * Primer)/(DNA + Primer))

where R is the gas constant (1.986 Cal/Mole*K).

Substituting G in the two equations and solving for T gives

T = ∆H/(∆S + R*Ln((DNA * Primer)/(DNA + Primer)))

assuming that the concentration of DNA and DNA-primer complex are equal.

It has been determined empirically that there is a 5 kcal free energy (3.4 kcal) (Sugimoto et al.
1996) change during the transition from single-stranded to B-form DNA. This is presumably helix
initiation energy. Finally, adding an adjustment for salt gives the equation that the Ta calculator
uses:

T = (∆H – 5(KCal/K*Mole))/(∆S + (R * ln(1/(primer)))) + 16.6 log10 (SaltMolarity)

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Chapter 5 Creating Protocols

No adjustment constant for salt concentration is needed, since the various parameters were
determined at 1 M NaCl, and the log10 of 1 is zero.

The thermodynamic calculations assume that annealing occurs at pH 7.0. The Tm calculations
assume that the sequences are not symmetrical and contain at least one G or C.

The oligonucleotide sequence should be at least 14 bases long to give reasonable Tm values.
Less than 14 bases uses the base pair counting method (see Table 7 that follows).

Table 7. Breslauer interaction constants

Interaction ∆H ∆S ∆G

AA TT 9.1 24 1.5

AT TA 8.6 23.9 1.5

AC TG 6.5 17.3 1.3

AG TC 7.8 20.8 1.6

TA AT 6 16.9 0.9

TT AA 9.1 24 1.9

TC AG 5.6 13.5 1.6

TG AC 5.8 12.9 1.9

CA GT 5.8 12.9 1.9

CT GA 7.8 20.8 1.6

CC GG 11 26.6 3.1

CG GC 11.9 27.8 3.6

GA CT 5.6 13.5 1.6

GT CA 6.5 17.3 1.3

GC CG 11.1 26.7 3.1

GG CC 11 26.6 3.1

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Using the T a Calculator

To use the Ta Calculator

1. To open the Ta Calculator, do one of the following:

n If you are currently in the Protocol AutoWriter, click Ta Calculator.

n In the Home window, select Tools > Ta Calculator.

The Ta Calculator dialog box appears.

2. In the Forward Primer text box, type or paste the forward primer sequence.

Tip: You can also use the A, T, G, C buttons on the left side of the dialog box to enter the
sequence.

3. Type or paste the reverse primer sequence in the Reverse Primer text box.

4. Click Calculate.

The Ta Calculator calculates and displays the Tm of each primer and the average Tm and Ta
values, for example:

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Chapter 5 Creating Protocols

If the primer Tm values are more than 4°C apart, the Protocol AutoWriter uses the lower primer
Tm value + 2°C as a basis for calculating the Ta value, which you can further modify by
changing the enzyme and reaction speed.

The Ta Calculator generates an annealing temperature for standard speed with iTaq DNA
polymerase. When using a different enzyme, the speed settings automatically adjust the Ta.

5. Do one of the following:

n If you opened the Ta Calculator from the Protocol AutoWriter, click OK. You return to the
Protocol AutoWriter. The annealing temperature is automatically modified.

n If you opened the Ta Calculator from the Tools menu, record the calculations and click
Cancel to close the calculator.

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Chapter 6 Preparing Plates
A plate file contains information about run parameters such as scan mode, fluorophores, and well
contents. After the run, CFX Maestro™ software links the well contents to the fluorescence data
collected during the run and applies the appropriate analysis in the Data Analysis window. For
example, wells loaded with standard sample type are used to generate a standard curve.

CFX Maestro software provides two options for creating plates: The Plate Editor for real-time PCR
runs and the Setup Wizard for normalized gene expression analysis.

The Plate Editor includes the following features:

n Standard fluorophores and sample types to assign to plate wells

n Ability to set reference target and control sample for gene expression analysis

n Ability to edit plate setup before, during, or after a run

n Ability to save plate files for reuse

n Ability to print the plate file to a default printer

The Setup Wizard guides you through creating a plate layout for normalized gene expression
analysis. You can use the Setup Wizard before, during, or after a run.

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Chapter 6 Preparing Plates

Plate Editor Window

You use the Plate Editor to create custom plates or modify existing plates.

LEGEND

1. The menu bar provides quick access to File and Settings menu commands as well as plate
editing tools options.

2. The toolbar provides quick access to important plate loading functions.

3. The main pane displays the plate outline and the plate options as you apply them.

4. The right pane displays options that you use to customize your plate.

5. The bottoms pane displays the plate type and provides quick access to viewing options.

File Menu Commands

Save — saves the plate data file in the location specified in the File tab in the User Preferences
dialog box. See Changing the Default File Settings on page 33 for more information.

Save As — saves the open plate data file with a new name that you supply.

Print — prints the open plate data file.

Close — closes the Plate Editor.

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Edit Menu Commands

Undo — reverts a change to a plate file until the plate file is saved.

Redo — reverses the most recent Undo action unless the plate file has been saved.

Settings Menu Commands

Plate Size — opens a dialog box from which you can select a plate size for the run.

Note: The plate size must be the same as the block size on the instrument on which the
run is performed. Choose 384-well for the CFX384 Touch™ system or 96-well for the
CFX96 Touch™, CFX96 Touch™ Deep Well, or CFX Connect™ system.

Plate Type — opens a dialog box from you can choose the type of wells in the plate that holds your
samples, including BR White and BR Clear. For accurate data analysis, the plate type selected
must be the same as the plate type used in the run.

Note: You must calibrate new plate types. See Calibrating New Dyes on page 52 for more
information.

Number Convention — opens a dialog box from which you can choose how to display units. The
default is to display units in scientific notation.

Units — opens a dialog box from which you can choose the units to show in the spreadsheets
when performing quantification of unknowns vs. a standard curve.

Editing Tools Menu Commands

Setup Wizard — opens the Setup Wizard, in which you can define layout and analysis parameters
for the current plate. You can use the Setup Wizard before, during, or after a run has completed.

Spreadsheet View/Importer — opens the View dialog box, which displays the plate layout as a
template in spreadsheet format. You can use this dialog box to export or import plate template data
in .csv format.

Flip Plate — flips the plate contents 180o.

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Chapter 6 Preparing Plates

Toolbar Commands

— reverts a change to a plate. CFX Maestro software supports up to undo actions.

— reverses the most recent Undo action. CFX Maestro software supports up to ten redo
actions.

— saves the current plate file.

— displays a dropdown list from which you can increase or decrease the
magnification of the plate view.

— displays a dropdown list from which you can select a scan

mode, which instructs the instrument from which channels to collect fluorescence data during a
run.

— opens the Well Groups Manager, which you can use to create well groups for
the current plate.

— displays a dialog box in which you can choose the colors and symbols for the
amplification traces.

— opens the View dialog box, which displays the plate layout as a
template in spreadsheet format. You can use this dialog box to export or import plate template data
in .csv format.

— opens the Setup Wizard, in which you can define the layout and analysis
parameters for the current plate. You can use the Setup Wizard before, during, or after a run.

— opens the Plate tab in the User Preferences dialog box, in which you can
define plate layout parameters and create or delete target, sample, and biological group names.
Changes that you make in the Plate tab are available the next time you open the Plate Editor.

— displays the necessary steps for setting up a plate and loading the wells.

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 Creating a Plate File Using the Plate Editor

Creating a Plate File Using the Plate Editor

Using the Plate Editor, you can create custom plate files. You can also edit and save previously
saved plate files or sample plate files shipped with CFX Maestro software.

To create a new plate file, perform the following:

n Open a plate file in the Plate Editor.

n Select the plate type.

Note: The plate type for the plate file must be the same as the plate in the reaction
module.

n Select the scan mode to use in the protocol.

n Select the fluorophores to use in the plate.

n Select the sample type, targets, and samples.

n Select technical replicates, if appropriate.

n Save the plate layout.

Tip: To create a new plate from previously saved or sample plate files, see Opening an
Existing Plate File in the Plate Editor on page 77.

Opening a New Plate File in the Plate Editor

CFX Maestro software offers multiple options to open a new plate file:

n From the Home window

n From the Startup Wizard dialog box

n From the Run Setup dialog box

To open a new plate file from the Home window

u Select File > New > Plate.

The Plate Editor window opens displaying the default plate file for the selected instrument.

Tip: For information about setting your default plate file, see Changing the Default File
Settings on page 33.

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Chapter 6 Preparing Plates

To open a new plate file from the Startup Wizard

1. In the Home window, do one of the following to open the Startup Wizard if it is not in view:

n Select View > Startup Wizard.

n Click Startup Wizard on the toolbar.

By default, the Startup Wizard displays the Run setup tab with the CFX96™ instrument
selected.

2. If necessary, select the instrument type from the dropdown list.

3. To create a new plate, click User-defined as the run type.

The Run Setup dialog box opens displaying the Protocol tab.

4. Click the Plate tab and click Create New.

The Plate Editor window opens displaying the default plate layout for the selected instrument.

To open a new plate file from the Run Setup dialog box

1. In the Home window, do one of the following to open the Run Setup dialog box:

n Select Run > User-defined Run.

n Click User-defined Run Setup on the toolbar.

The Run Setup dialog box opens to the Protocol tab.

2. To create a new plate, click the Plate tab and click Create New.

The Plate Editor window opens displaying the default plate layout for the selected instrument.

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 Creating a Plate File Using the Plate Editor

Opening an Existing Plate File in the Plate Editor

CFX Maestro software provides sample plate files that you can edit and save as a new plate. You
can also create a new plate file from a previously saved plate file.

To open a sample plate file

1. In the Home window, select File > Open > Plate.

Windows Explorer opens to the location of the CFX Maestro Sample files folder.

2. Open the Sample files folder, and then open the Plates folder.

3. Select the plate of choice and click Open.

The sample plate file opens in the Plate Editor window.

4. Select File > Save As and save the plate file with a new name or in a new folder.

To open a previously saved plate file

1. In the Home window, do one of the following:

n Select File > Open > Plate, navigate to and select the target plate, and click Open.

n Open the Startup Wizard and do one of the following:

o To edit an existing plate file, click Select Existing and navigate to the target file.

o To edit the displayed plate file, click Edit Selected.

The target plate opens in the Plate Editor window.

2. Select File > Save As and save the plate file with a new name or in a new folder.

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Chapter 6 Preparing Plates

Setting Up a New Plate File

Tip: If your plate file includes the required parameters (for example, if you are editing a sample
or existing plate file) you can skip this section. Proceed to Assigning Optional Parameters to
the Plate File on page 84.

New plate files require the following parameters:

n Plate size

n Plate type

n Scan mode

n One fluorophore (dye)

n One sample type

Selecting the Plate Size and Type

Important: You must select a plate size during plate setup. You cannot change the plate size
during or after a run.

The software applies the plate size and type to all the wells during the run. Ensure that the plate
size selected is the same as the plate you will use in the run.

Bio-Rad’s CFX96 Touch, CFX96 Touch Deep Well, CFX Connect, and CFX384 Touch instruments
are factory-calibrated for many fluorescent dye and plate combinations. Calibration is specific to
the instrument, dye, and plate type. Ensure that the fluorophore you plan to use is calibrated for the
plate type you select.

Tip: To calibrate a new combination of dye and plate type on an instrument, select
Tools > Calibration Wizard. For information about calibrating dyes and plate types, see
Calibrating New Dyes on page 46.

Selecting Scan Mode

The CFX96 Touch and CFX96 Touch Deep Well systems excite and detect fluorophores in six
channels. The CFX Connect system excites and detects fluorophores in three channels. The
CFX384 Touch system excites and detects fluorophores in five channels. All systems use multiple
data acquisition scan modes to collect fluorescence data during a run.

CFX Maestro software provides three scan modes:

n All Channels

o Scans channels 1 through 5 on the CFX96 Touch and CFX96 Touch Deep Well systems

o Scans channels 1 and 2 on the CFX Connect system

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 Creating a Plate File Using the Plate Editor

o Scans channels 1 through 4 on the CFX384 Touch system

n SYBR® /FAM

o Scans only channel 1

o Provides a fast scan

n FRET

o Scans only the FRET channel

o Provides a fast scan

Selecting Fluorophores

Important: Before beginning the run, CFX Maestro software verifies that the fluorophores you
specified in the plate are calibrated on that instrument. You cannot run a plate if it includes
fluorophores that have not been calibrated on that instrument.

You must load at least one fluorophore to the plate layout before the run. You can add as many
fluorophores as necessary at this time but the plate must contain at least one fluorophore. The
selected fluorophores appear as options for targets in Target Names.

You use the Select Fluorophores dialog box to load fluorophores (or plate dyes) into the Plate
Editor well loading controls. The fluorophores that appear in the Select Fluorophores dialog box
depend on the scan mode you select:

n All Channels

All available fluorophores appear.

Tip: You can add as many fluorophores as necessary, but you can load only one
fluorophore per channel in each well.

n SYBR®/FAM

Only channel 1 fluorophores appear.

n FRET

Only the channel 6 fluorophore appears.

Tip: The channel 6 FRET fluorophore appears only when FRET is the selected scan
mode. It is not available for All Channels scan mode.

Note: You cannot directly add fluorophores to or remove them from the Select Fluorophore
dialog box. You must calibrate new fluorophores on an instrument using the Calibration
Wizard. After calibration, the new fluorophore is automatically added to this list. For more
information, see Calibrating New Dyes on page 46.

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Chapter 6 Preparing Plates

Selecting Sample Types

Important: You must select at least one sample type to assign to the plate wells before the
run.

CFX Maestro software offers five sample types:

n Unknown

n Standard

n NTC (no template control)

n Positive Control

n Negative Control

n NRT (no reverse transcriptase)

You assign the sample types to the plate wells.

Setting Up a New Plate

To set up a new plate

1. Open a new plate in the Plate Editor window.

2. To set the plate size, select Settings > Plate Size and select the appropriate plate size from the
dropdown menu.

3. To set the plate type, select Settings > Plate Type and select either BR White or BR Clear from
the dropdown menu.

4. Optionally, from the Settings menu you can change the number convention and the display
units:

n To change the number convention, select Settings > Number Convention and select
Scientific Notation.

Tip: Scientific Notation is selected by default. In this case, selecting Scientific Notation
clears the default and sets the number convention to standard form.

n To change the display units, select Settings > Units and select a new unit value.

5. To set the scan mode, select the appropriate scan mode from the Scan Mode dropdown list in
the Plate Editor window toolbar.

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 Creating a Plate File Using the Plate Editor

6. Select the requisite flourophores for the plate:

a. In the right pane, click Select Fluorophores.

The Select Fluorophores dialog box appears. You see the fluorophores available for the
type of scan mode you selected in Step 5, for example:

b. To select a fluorophore, click its Selected checkbox.

Tip: To remove a fluorophore from the list, clear its Selected checkbox.

c. To change the display color of the fluorophore, click its Color box.

Note: The color you select represents the fluorophore in both the Plate Editor window
and the Data Analysis charts.

d. In the Color dialog box, select the color that you want or click Define Custom Colors and
create a new color to represent the fluorophore.

e. Click OK to save the changes and exit the Select Fluorophores dialog box.

7. You must select at least one well in which to load a sample type. By default, well A1 is
selected.

In the plate pane, do one of the following:

n To load multiple adjacent wells, click a well and drag to the target well.

n To load multiple nonadjacent wells, hold the Control key and click each well.

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Chapter 6 Preparing Plates

n To load an entire column with the same sample type, click the column number.

n To load an entire row, click its row number.

n To load the whole plate, click the upper left corner of the plate.

For example:

8. Assign a sample type to the selected well or wells from the Sample Type dropdown menu.

9. Assign at least one fluorophore to all wells that contain a sample type. You can assign more
than one fluorophore to a well or group of wells.

Note: You can assign only one fluorophore per channel. You cannot assign more than
one fluorophore from the same channel to the same well.

Tip: You can associate a target with the fluorophore or you can assign only the
fluorophore to the well at this time and associate a target to the fluorophore after you run
the experiment.

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n To assign only a fluorophore to the selected wells, in the Target Names section in the right
pane select the Load checkbox for the specific fluorophore.

n To associate a target with a fluorophore, in the Target Names section select a target name
from the dropdown list for the specific fluorophore. The software automatically selects its
Load checkbox.

10. For wells containing a Standard sample type, you must load a concentration. Each well can
have a different concentration value. By default, CFX Maestro software loads a concentration
of 1.00E+06 to all wells with a Standard sample type. You can change the value if necessary.

a. In the plate pane, select a Standard well or group of wells.

b. In the Concentration section click Load to load the value to the selected well or wells.

c. (Optional) To load another concentration, type the new value in the Concentration text box
and press enter.

d. Perform this step for all wells with sample type Standard.

Tip: To load the same concentration to all Standard wells, ensure that <All> appears
in the dropdown list below the Concentration value. To load the same concentration
value to all wells with a specific fluorophore, click the dropdown list and select the
fluorophore.

11. Click OK to save the new plate.

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Chapter 6 Preparing Plates

Assigning Optional Parameters to the Plate File

A plate file contains information about the contents of each well loaded with sample for a run. After
the run, CFX Maestro software links the well contents to the fluorescence data collected during the
protocol and applies the appropriate analysis in the Data Analysis window.

In CFX Maestro, you can assign parameters to each well in your plate before, during, or even after
you run experiments. You can assign the parameters to an existing plate file or to a new plate file.
These parameters include:

n Target names — the target or targets of interest (genes or sequences) in each loaded well.

n Sample names — the identifier or condition that corresponds to the sample in each loaded
well, such as mouse1, mouse2, or mouse3.

n Biological groups — the identifier or condition that corresponds to a group of wells, such as
0Hr, 1Hr, or 2Hr.

Tip: Target names, sample names, and biological groups must be the same between
wells to compare data in the Gene Expression tab of the Data Analysis window. Each
name must contain the same capitalization, punctuation, and spacing. For example,
“Actin” is not the same as “actin,” “2Hr” is not the same as “2 hr.,” and “Mouse 1” is not the
same as “mouse1.” To ensure naming consistency, enter the names in the Libraries
section in User > User Preferences > Plate, available on the Home window.

n Technical replicates — each well that is used to analyze the same combination of sample
and target(s); that is, replicate qPCR reactions.

n Dilution series — the amount to change the concentration of the Standard sample type within
a replicate group to create standard curve data to analyze.

Assigning a Target to Wells

Tip: You can assign the same target name to a single or multiple wells. You can also assign
multiple targets to the same well.

Important: Clicking OK after you assign a target saves the changes and disables Undo on the
Plate Editor toolbar. Take care when clicking OK.

To assign a target to a well or group of wells

1. In the Plate Editor, ensure that the well or group of wells have been assigned a sample type.

See Selecting Sample Types on page 80 for information about assigning sample types to
wells.

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 Assigning Optional Parameters to the Plate File

2. In the plate pane, select the well or group of wells:

n To select a single well, click the well.

n To select multiple adjacent wells, click a well and drag to the target well.

n To select multiple nonadjacent wells, hold the Control key and click each well.

n To select an entire column with the same sample type, click the column number.

n To select an entire row, click its row number.

3. In the right pane, select a name from the Target Name dropdown list for each selected
fluorophore.

4. Repeat Step 3 for each well or group of wells to which you must assign a target.

Tip: You can assign the same or a different target name for each selected fluorophore.

5. Click OK to accept the changes and save the plate.

Note: If you changed the plate in error, click Undo on the Plate Editor toolbar before you
click OK to accept the changes.

To remove a target name

u To remove a target name from the selected well or group of wells, clear its Load checkbox.

Important: Removing a target name from a well also removes its associated fluorophore. Take
care when removing a target name from a well.

To add a target name to the list

u To add a target name to the dropdown list, do one of the following:

n Type a name in the Target Name dropdown list and press Enter.

Tip: Target names that you add to one list appear in all other target lists.

n Click the green + symbol to the right of the dropdown list, type a name for the target and
press Enter.

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Chapter 6 Preparing Plates

n Click User Preferences on the toolbar and add the name to the Target Names library in
the Plate tab.

Important: Target names that you add in the dropdown list are available only for the
current plate, and only if you assign the name to a well and save the plate layout. If you do
not assign the name to a well and save the plate layout, the name is not saved and is not
available for future use. To permanently add a target name, also add it to the Target
Names library using the User Preferences dialog box. Names that you add to the library
are available after you open the Plate Editor again. See Setting Default Plate Parameters
on page 35 for more information.

To delete a target name from the list

1. Click User Preferences on the toolbar.

The User Preferences dialog box appears, displaying the Plate tab.

2. In the Target Names library in the Plate tab, select the name to delete and press the Delete
key.

3. Click OK to save changes and exit the User Preferences dialog box.

Important: You cannot delete target names that you saved with a plate file. Custom
names that you add to the Target Names dropdown list and do not use and save with the
plate are automatically removed from the list. Names that you delete from the Target
Names Library are permanently removed from the software and are no longer available to
users. Take care when deleting target names.

Assigning a Sample Name to Wells

Note: To assign a sample name, you must assign the selected wells at least one fluorophore.
If the selected wells are not assigned a fluorophore, the Sample Names dropdown list is
disabled. See Assigning a Target to Wells on page 84 for information about assigning
fluorophores.

Tip: You can assign only one sample name to each well or group of wells.

To assign a sample name to a well or group of wells

1. In the Plate Editor, ensure that the well or group of wells has been assigned a fluorophore.

2. In the plate pane, select the well or group of wells.

3. In the right pane, select a name in the Sample Names dropdown list.

The software automatically selects its Load checkbox.

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4. Repeat Step 3 for each well or group of wells to which you must assign a sample name.

5. Click OK to accept the changes and save the plate.

Note: If you changed the plate in error, click Undo on the Plate Editor toolbar before you
click OK to accept the changes.

To remove a sample name

u To remove a sample name from a selected well or group of wells, clear its Load checkbox.

To add a sample name to the list

u To add a sample name to the dropdown list, do one of the following:

n Type a name in the Sample Names dropdown list and press Enter.

n Click the green + symbol to the right of the dropdown list and type a name for the sample.

n Click User Preferences on the toolbar and add the name to the Sample Names library in
the Plate tab.

Important: Sample names that you add in the dropdown list are available only for the
current plate, and only if you assign the name to a well and save the plate layout. If you do
not assign the name to a well and save the plate layout, the name is not saved and is not
available for future use. To permanently add a sample name, also add it to the Sample
Names library using the User Preferences dialog box. Names that you add to the library
are available after you open the Plate Editor again. See Setting Default Plate Parameters
on page 35 for more information.

To delete a sample name from the list

1. Click User Preferences on the toolbar.

The User Preferences dialog box appears, displaying the Plate tab.

2. In the Sample Names library in the Plate tab, select the name to delete and press the Delete
key.

3. Click OK to save changes and exit the User Preferences dialog box.

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Chapter 6 Preparing Plates

Important: You cannot delete sample names that you have saved with a plate file.
Custom names that you add to the Sample Names list and do not use and save with the
plate are automatically removed from the dropdown list. Names that you delete from the
Sample Names Library are removed from the software and are no longer available to
users. Take care when deleting sample names.

Assigning Biological Groups to Wells

Note: To assign a biological group, you must assign the selected wells at least one
fluorophore. Assigning a fluorophore enables the Biological Groups dropdown list. See
Assigning a Target to Wells on page 84 for information about assigning fluorophores.

Tip: You can assign one biological group to each well or group of wells.

To assign a biological group to a well or group of wells

1. In the Plate Editor, ensure that the well or group of wells has been assigned a fluorophore.

2. In the plate pane, select the well or group of wells.

3. In the right pane, select a name in the Biological Group dropdown list.

CFX Maestro software automatically selects its Load checkbox.

4. Repeat Step 3 for each well or group of wells to which you must assign a biological group.

5. Click OK to accept the changes and save the plate.

Note: If you changed the plate in error, click Undo on the Plate Editor toolbar before you
click OK to accept the changes.

Tip: Assigning biological group names to wells enables Biological Group Analysis
Options in the Experiment Settings dialog box, in which you can perform sample analysis
in one of four configurations. SeeChanging Experiment Settings on page 95 for more
information.

To remove a biological group

u To remove a biological group from the selected well or group of wells, clear its Load checkbox.

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 Assigning Optional Parameters to the Plate File

To add a biological group name to the list

u To add a biological group name to the dropdown list, do one of the following:

n Type a name in the Biological Group dropdown box and press Enter.

n Click the green + symbol to the right of the dropdown list and type a name for the
biological group.

n Click User Preferences on the toolbar and add the name to the Biological Names library
in the Plate tab.

Important: Biological group names that you add in the dropdown list are available only
for the current plate, and only if you assign the name to a well and save the plate layout. If
you do not assign the name to a well and save the plate layout, the name is not saved and
is not available for future use. To permanently add a biological group name, also add it to
the Biological Names library using the User Preferences dialog box. Names that you add
to the library are available after you open the Plate Editor again. See Setting Default Plate
Parameters on page 35 for more information.

To delete a biological group name from the list

1. Click User Preferences on the toolbar.

The User Preferences dialog box appears, displaying the Plate tab.

2. In the Biological Names library in the Plate tab, select the name to delete and press the Delete
key.

3. Click OK to save changes and exit the User Preferences dialog box.

Important: You cannot delete biological group names that you saved with a plate file.
Custom names that you add to the Biological Group Names dropdown list and do not use
and save with the plate are automatically removed from the list. Names that you delete
from the Biological Names Library are permanently removed from the software and are no
longer available to users. Take care when deleting biological names.

To view all biological groups on the plate

u Click Show Biological Groups to view all biological groups on the plate.

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Chapter 6 Preparing Plates

Each group is identified by a specific color and the Show Biological Groups button changes to
Hide Biological Groups.

Click Hide Biological Groups to clear the color in the wells. Alternatively, you can click any well
in the plate to hide biological groups.

Assigning Technical Replicate Numbers to Wells

Important: To assign technical replicate numbers, the selected wells must contain identical
well contents. That is, the selected wells must have the same sample type and fluorophore. If
appropriate, they must also be assigned the same target and sample names and the same
biological group. If they are not the same, CFX Maestro software does not enable this option.

To assign technical replicate numbers to a group of wells

1. In the Plate Editor, ensure that the contents of the group of wells are identical.

2. In the plate pane, select the target group of wells.

3. To assign the same replicate number to all selected wells, in the Replicate # section in the
right pane type the replicate number in the box and select Load.

4. (Optional) To apply a replicate series to a set of selected wells:

a. Click Technical Replicates. The Replicate # section expands to display the following
options:

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 Assigning Optional Parameters to the Plate File

n Replicate size — a number that represents the number of wells in each group of
replicates

n Starting replicate # — the first number in the replicate series for the selected group
of replicates

Note: By default, CFX Maestro software displays the starting replicate number as one
number greater than the last technical replicate number assigned in the plate. For
example, if the last technical replicate number in the plate is five, the next starting
number is six. You can change the starting number to any number that is not already
assigned.

n Loading direction (Horizontal or Vertical)

b. Click Apply to apply the parameters to the series and return to the Replicate # display.

5. Click OK to accept the changes and save the plate.

Note: If you changed the plate in error, click Undo on the Plate Editor toolbar before you
click OK to accept the changes.

To remove a well from a replicate series

u Select the well or group of wells to be removed and clear the Replicate # Load checkbox.

Alternatively, you can click Clear Replicate # to clear the replicate number from a selected well
or group of wells.

To view all technical replicates on the plate

u Click Show Technical Replicates to view all technical replicates on the plate.

Each group is identified by a specific color and the Show Technical Replicates button changes
to Hide Technical Replicates.

Click Hide Technical Replicates to clear the color in the wells. Alternatively, you can click any
well in the plate to hide technical replicates.

Assigning a Dilution Series to Standard Sample Types

As previously mentioned, all wells with the sample type Standard must be assigned a
concentration value. You can assign a dilution series to multiple wells with the sample type
Standard.

Note: In order to assign a dilution series to a group of wells, the wells must be included in a
technical replicate series. See Assigning Technical Replicate Numbers to Wells on page 90
for information about adding wells to a replicate series.

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Chapter 6 Preparing Plates

To assign a dilution series to a group of Standard sample wells

1. In the Plate Editor, ensure the following requirements are met:

n The sample type for the group of wells is Standard.

n All wells in the group are assigned at least one fluorophore and they all contain the same
fluorophores.

n All wells in the group are included in the same technical replicate series.

Note: CFX Maestro software enables the Dilution Series option only when all
selected wells meet these criteria.

2. In the plate pane, select the target group of wells.

3. In the Concentration section in the right pane, click Dilution Series. The Concentration section
expands to display the following options:

n Starting concentration — the concentration value from which the series starts

n Replicates from and to — the replicates in the series to which the dilution factor will be
applied

n Dilution factor — the amount to change the concentration within each replicate group

4. Set the values for the options or accept the defaults.

5. By default, the dilution series decreases by the dilution factor. Select Increasing to increase
the dilution series.

6. (Optional) By default, the dilution factor applies to all fluorophores in the replicate series. If
your series contains more than one fluorophore and you want to apply the dilution to a single
fluorophore, select it from the dropdown list.

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 Assigning Optional Parameters to the Plate File

7. Click Apply to apply the dilution series to the group of wells and return to the Concentration
view.

8. Click OK to accept the changes and save the plate.

Note: If you changed the plate in error, click Undo on the Plate Editor toolbar before you
click OK to accept the changes.

Copying Well Contents into Another Well

You can copy the contents of a well and paste it into a single or multiple wells. However, you can
copy the contents of only a single well. You cannot select multiple wells and copy their contents.

To copy well contents into another well

1. In the plate pane, select the well to be copied.

2. Right-click the well and select Copy Well.

3. Select the well or wells into which content is to be pasted:

n To select a single well, click on the well.

n To select multiple adjacent wells, click a well and drag to the target well.

n To select multiple nonadjacent wells, hold the Control key and click each well.

4. With the target wells selected, right-click and select Paste Well.

CFX Maestro software pastes the contents of the first well into the selected wells.

Adding a Note to a Well

You can add a descriptive note to a well. You can view the well notes in the Quantification tab in
the Data Analysis window.

To add a note to a well

1. In the plate pane, select the well or wells to which you plan to add a note.

2. In the View section in the bottom pane, select Well Note.

The Well Note dialog box appears in the right pane.

3. Type the content for the note in the textbox and press Enter.

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Chapter 6 Preparing Plates

The text appears at the bottom of the selected wells.

Tip: If you created a previous well note, you can select it from the dropdown list and apply
it to the selected wells.

Clearing Wells of All Content

You can clear an individual well, a group of wells, or the whole plate of all content. Clearing wells
does not remove the fluorescence data collected during the plate read.

Clearing a well permanently removes the content from the well. If you click OK and save the plate
after clearing a well, you cannot undo the clear action. Take care when clearing wells.

To clear wells of all settings

1. In the Plate Editor, select the well or group of wells in the plate pane:

n To select a single well, click on the well.

n To select multiple adjacent wells, click a well and drag to the target well.

n To select multiple nonadjacent wells, hold the Control key and click each well.

n To select an entire column with the same sample type, click the column number.

n To select an entire row, click its row number.

2. In the right pane, click Clear Wells.

CFX Maestro software clears the selected wells of all settings.

3. Do one of the following:

n If you cleared the wells in error, click Undo on the Plate Editor toolbar before you click OK
to accept the changes.

Important: Clicking OK before you click Undo saves the changes and disables Undo
on the Plate Editor toolbar.

n Click OK to accept the changes and save the plate.

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 Changing Experiment Settings

Changing Experiment Settings

Use the Experiment Settings dialog box to view or change the list of targets, samples, or biological
groups, or to set the gene expression analysis sample group to analyze if you assigned biological
groups to wells in the plate.

In the Experiment Settings dialog box, the Targets tab displays a list of target names for each PCR
reaction, such as the target gene or gene sequences of interest.

The Samples and Biological Groups tab displays a list of sample and biological group names that
indicate the source of the target, such as a sample taken at 1 hour (1Hr) or from a specific
individual (mouse1).

To change plate settings using the Experiment Settings dialog box

1. To open the Experiment Settings dialog box, do one of the following:

n In the right pane in the Plate Editor, click Experiment Settings.

n In the Gene Expression tab in the Data Analysis window, click Experiment Settings.

The Experiment Settings dialog box appears displaying the contents of the Targets tab.

2. To add a new target, sample, or biological group name, in the appropriate tab type a name in
the New textbox and click Add.

3. To remove one or more target, sample, or biological group names from the list, in the
appropriate tab select the item’s checkbox in the Select to Remove column and click Remove
checked item(s).

4. CFX Maestro software excludes the sample type NTC (no template control) from gene
expression analysis.

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To include NTC sample types, clear its checkbox in the Exclude the following sample types
section. You can choose to exclude the following sample types by selecting the appropriate
checkbox:

n NRT (no reverse transcriptase)

n Negative Control

n Positive Control

n Standard

5. In the Targets tab:

a. To select a target as the reference for gene expression data analysis, select it in the
Reference column.

b. To hide analysis settings that will be applied in the Gene Expression tab in the Analysis
Settings window, clear Show Analysis Settings.

The software hides the following columns:

n Color

n Show Chart

n Auto Efficiency

n Efficiency (%)

c. To change the color of the target as it is graphed in the Gene Expression chart, click its
cell in the Color column, select a new color in the Color dialog box that appears, and click
OK.

d. To display the target in the selected color in the Gene Expression chart, select its
checkbox in the Show Graph column.

e. By default, CFX Maestro automatically calculates the relative efficiency for a target if its
data include a standard curve.

To use a previously determined efficiency value, type the value in its cell in the Efficiency
(%) column and press the Enter key. CFX Maestro clears the Auto Efficiency checkbox.

6. In the Samples and Biological Groups tab:

a. To select a sample or biological group as the control sample for gene expression data
analysis, select its checkbox in the Control column.

b. To assign the control condition to a sample or biological group for a run, click its checkbox
in the Control column.

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 Creating Well Groups

c. If it is not already selected, click Show Analysis Settings to view or change analysis
parameters that will be applied in the Gene Expression tab. The software hides the Color
and Show Chart columns.

7. Click OK to save the parameters in the Experiment Settings dialog box and return to the Plate
Editor window.

Creating Well Groups

Well groups divide a single plate into subsets of wells that can be analyzed independently in the
Data Analysis window. Once well groups are set up, select one in the Data Analysis window to
analyze the data as an independent group. For example, set up well groups to analyze multiple
experiments run in one plate or to analyze each well group with a different standard curve.

Note: The default well group is All Wells.

To create well groups

1. To open Well Groups Manager, do one of the following:

n In the Plate Editor toolbar, click Well Groups.

n In the Data Analysis window, click Manage Well Groups.

The Well Groups Manager dialog box appears.

2. Click Add to create a new group. The dropdown menu displays the group name as Group 1 for
the first group.

3. Select the wells for the well group in the plate view by clicking and dragging across the group
of wells. Selected wells appear blue in the Manager.

4. (Optional) To change the name of the group, select its name in the dropdown menu and type a
new name.

5. (Optional) To delete a well group, select its name in the dropdown list and click the Delete.

6. Click OK to finish and close the window, or click Cancel to close the window without making
changes.

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Right-Click Menu Items for the Well Groups Manager Dialog Box

Table 8 lists the menu items available in the Well Groups Manager dialog box when you right-click
on any well.

Table 8. Right-click menu items in the Plate Editor Well Selector dialog box

Item Function
Copy Copies the well contents, which can then be pasted into
another well or wells.

Copy as Image Copies the well selector view as an image.

Print Prints the well selector view.

Print Selection Prints only the selected cells.

Export to Excel Exports the data to an Excel spreadsheet.

Export to Csv Exports the data as a comma-separated document.

Export to Xml Exports the data as an .xml document.

Export to Html Exports the data as an .html document.

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 Changing Trace Styles

Changing Trace Styles

During plate setup and while a run is in progress, you can modify the color and style of the
amplification traces. You can then easily view the traces in the real-time status window as the data
are being collected.

To change trace styles

1. Click Trace Styles in the Plate Editor toolbar.

The Trace Styles dialog box appears for the open plate, for example:

2. To display the trace styles by a specific fluorophore, select it from the Fluorophores dropdown.

3. To change the trace display:

a. Select the trace type from the Wells dropdown list.

b. Click its color in the Color column.

c. In the Color dialog box that appears, choose another color for the trace and click OK.

The change for the well type appears in the grid below.

d. (Optional) Select a symbol for the trace from the Symbols dropdown list.

4. To quickly change the color set, click the appropriate choice in the Color Quick Set section.

5. To view well labels in the grid, select the label type in the Well Labels section.

6. Click OK to save changes or Cancel to cancel changes.

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Chapter 6 Preparing Plates

Viewing the Plate in Spreadsheet Format

The Spreadsheet View/Importer tool displays the contents of a plate in spreadsheet format. You
can use the Spreadsheet View/Importer tool to export well contents in tab-delimited format to an
application such as Microsoft Excel. You can also import well contents from a tab-delimited
application.

To use the Spreadsheet View/Importer tool

1. On the Plate Editor toolbar, click Spreadsheet View/Importer to open the Plate Spreadsheet
View dialog box.

2. The Spreadsheet View dialog box displays the contents in the plate for a single fluorophore.
To view the plate contents for another fluorophore, select it from the Fluors List dropdown list.

3. Click Export Template to export a template of the plate spreadsheet to an Excel file (.csv
format). You can edit this template to import well content information.

4. (Optional) Click Import to import well contents from a comma-delimited file.

5. To sort the spreadsheet according to the data in a specific column, click the triangle next to a
column name.

Tip: You can edit the contents of any cell in a column that has an asterisk (*) beside the
column name (for example *Target Name).

Note: Select the units for the standard curve data in the Quantity column by opening the Plate
Editor and selecting Settings > Units in the menu bar. After the plate run is completed, the data

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 Viewing the Plate in Spreadsheet Format

from these standards appear in the Standard Curve chart in the Quantification tab in the Data
Analysis window with the units you select.

Right-Click Menu Items for the Plate Spreadsheet View/Importer Tool

Table 9 lists the menu items available in the Spreadsheet View/Importer tool when you any right-
click on any well in the tool.

Table 9. Right-click menu items in the Plate Spreadsheet View/Importer tool

Item Function
Copy Copies the entire spreadsheet.

Copy as Image Copies the spreadsheet as an image file.

Print Prints the spreadsheet.

Print Selection Prints only the selected cells.

Export to Excel Exports the file to an Excel spreadsheet.

Export to Text Exports the file as a text file.

Export to Xml Exports the file as an .xml file.

Export to Html Exports the file as an .html file.

Find Searches for specific text.

Sort Sorts the spreadsheet by selecting up to three columns of data in

the Sort window.

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Chapter 6 Preparing Plates

Creating a Plate Layout Using the Plate Setup Wizard

You can use the Setup Wizard to enter the plate layout information that is needed for normalized
gene expression analysis, including:

n Target names

n Sample names

n Location of targets and sample on the plate

n Reference gene(s)

n Control sample

You can use the Setup Wizard before, during, or after a run.

Using the Plate Setup Wizard

This section explains how to create a plate layout using the plate Setup Wizard. To view the
contents of each well in the plate more easily, click Zoom plate at the top of the Setup Wizard.

Important: Returning to the Auto layout tab while on any other tab in the Setup Wizard resets
the plate layout. Take care when selecting this tab.

Tip: You can reset the layout by selecting Tools > Clear Plate in the Setup Wizard.

To use the plate Setup Wizard

1. Open the Plate Editor.

2. To open the Setup Wizard, do one of the following:

n Choose Editing Tools > Setup Wizard.

n Click Setup Wizard on the Plate Editor toolbar.

The Setup Wizard appears displaying the Auto layout tab.

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 Creating a Plate Layout Using the Plate Setup Wizard

3. In the Auto layout tab, do the following:

a. Click a well in the grid and drag across and down to specify the area on the plate in which
you plan to load sample.

b. Enter the number of targets and samples to load.

Tip: The number of targets and samples must equal the number of selected cells. If
the numbers entered do not fit in the area selected, modify the numbers or plate
selection area. The orientation of items on the plate and their grouping can be
specified.

c. (Optional) Change the plate orientation. For example, you can set targets in columns and
samples in rows, or group by samples.

d. Click Next to proceed to the Target names tab.

Note: If your plate layout does not have a regular pattern, use the Target names tab to
manually position your targets or the Sample names tab to manually position your
samples on the plate. Click and drag to select multiple wells.

4. In the Target names tab, define target names for the target groups:

a. Do one of the following:

n To rename targets by group, set Select by to Target.

n To rename targets by well, set Select by to Well.

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b. Select a target group or well in the grid and type a name in the Target name dropdown
list.

Tip: Press Tab to select the next group or well to the right or Enter to select the next
group or well below. Alternatively, on the Target name and Sample name tabs, hold
the Control key and click a well to select multiple wells that are not adjacent.

c. Click Next to proceed to the Sample names tab.

5. In the Sample names tab, define sample names for the sample groups.

6. Click Next to proceed to the Reference targets tab.

7. In the Reference targets tab, select one or more targets to use as references for normalized
gene expression and click Next to proceed to the Control sample tab.

8. In the Control sample tab, select one sample to use as a control for relative gene expression
calculations.

9. Click OK to save the plate layout and return to the Plate Editor, in which you can further define
plate parameters. See Assigning Optional Parameters to the Plate File on page 84 for more
information.

Alternatively, click Previous to return to a previous tab to make any changes.

Note: Returning to the Auto layout tab automatically resets the plate. Take care when
clicking Previous.

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Chapter 7 Running Experiments
This chapter explains how to run custom (user-defined) or PrimePCR™ assay experiments using
CFX Maestro™ software.

A run data file contains the protocol and plate information for the run. The file also contains the
data from the analyses that CFX Maestro performs after the run completes.

CFX Maestro software makes it easy to set up and run user-defined or PrimePCR experiments.
The Run Setup window guides you through the common steps of setting up an experiment, leading
you to the Start Run dialog box, from which you start the run.

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Chapter 7 Running Experiments

The Run Setup Window

The Run Setup window provides quick access to the files and settings needed to set up and run an
experiment. When you choose to run a user-defined experiment, the Run Setup window opens
displaying the Protocol tab. When you choose to run a PrimePCR experiment, the Run Setup
window opens displaying the Start run tab.

Tip: See Performing PrimePCR Experiments on page 120 for information about PrimePCR;
see Start Run Tab on page 112 for information about the Start Run tab.

LEGEND

1. Tabs guide you through setting up and running an experiment:

n Protocol tab — select an existing protocol to run or edit, or to create a new
protocol in the Protocol Editor.
n Plate tab — select an existing plate to run or edit, or to create a new plate in the
Plate Editor.
n Start Run tab — view the experiment settings, select one or more instrument
blocks, and begin the run.

2. The main window displays the options for each tab as you apply them.

3. Navigation buttons lead you to the Start Run tab.

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Accessing the Run Setup Window

To access the Run Setup window

u Do one of the following:

n In the Run setup tab in the Startup Wizard, click either User-defined or PrimePCR.

n In the Home window, click either User-defined Run Setup or PrimePCR Run Setup on the
toolbar.

n In the Home window, select either Run > User-defined Run or Run > PrimePCR Run.

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Chapter 7 Running Experiments

Protocol Tab
The Protocol tab displays a preview of the protocol file that you plan to run. A protocol file contains
the instructions for the instrument temperature steps as well as instrument options that control the
ramp rate, sample volume, and lid temperature.

By default, the software displays the protocol defined in the File Selection for Run Setup section in
the Files tab in the User > User Preferences dialog box. You can change the default protocol in the
User Preferences dialog box. See Changing the Default File Settings on page 33 for more
information.

In the Protocol tab, you can:

n Create a new protocol to run

n Select an existing protocol to run or edit

For more information about creating and modifying protocols, see Chapter 5, Creating Protocols.

To create a new protocol

1. On the Protocol tab, click Create New.

The Protocol Editor appears.

2. Use the Protocol Editor to create a new protocol.

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 Protocol Tab

3. Click OK to save the protocol and return to the Protocol tab in Run Setup.

4. View the details of the protocol and do one of the following:

n If the details are correct, click Next to proceed to the Plate tab.

n If the details are incorrect, click Edit Selected to return to the Protocol Editor window.
Revise the protocol, save changes, and then click Next on the Protocol tab to proceed to
the Plate tab.

To select an existing protocol

1. On the Protocol tab, do one of the following:

n Click Select Existing and navigate to an existing protocol.

n Click Express Load and select a protocol from the dropdown list of protocols.

Tip: You can add protocols to or remove them from the Express Load dropdown list.
See Adding and Removing Express Load Protocols that follows for more information.

2. View the details of the protocol and do one of the following:

n If the details are correct, click Next to proceed to the Plate tab.

n If the details are incorrect, click Edit Selected to open the Protocol Editor. Revise the
protocol, save changes, and then click Next on the Protocol tab to proceed to the Plate
tab.

Adding and Removing Express Load Protocols

You can modify the contents of the Express Load dropdown list that appears in the Protocol Editor.
The protocols in this list are saved in the following folder:

c:\Users\Public\Documents\Bio-Rad\CFX\Users\<user_name>\ExpressLoad\

To modify the Express Load list of protocols

1. Navigate to and open the ExpressLoad folder.

2. Review the protocol files (.pcrl) in the folder.

3. Do either of the following:

n Delete protocols from the folder to remove them from the dropdown list.

n Copy protocols into the folder to add them to the dropdown list.

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Chapter 7 Running Experiments

Plate Tab
Note: If the protocol selected in the Protocol tab does not include a plate read step for real-
time PCR analysis, the Plate tab is hidden. To view the Plate tab, add at least one plate read to
the protocol.

The Plate tab displays a preview of the plate file you plan to load. In a real-time PCR run, the plate
file contains a description of the contents of each well including its fluorophores, the scan mode,
and the plate type. CFX Maestro software uses these descriptions for data collection and analysis.

By default, the software displays the plate defined in the File Selection for Run Setup section in the
Files tab in the User > User Preferences dialog box. You can change the default plate in the User
Preferences dialog box. See Changing the Default File Settings on page 33 for more information.

In the Plate tab, you can:

n Create a new plate to load.

n Select an existing plate to load or edit.

For more information about creating and modifying plates, see Chapter 6, Preparing Plates.

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 Plate Tab

To create a new plate

1. On the Plate tab, click Create New.

The Plate Editor appears.

2. Use the Plate Editor to create a new plate.

3. Click OK to save the plate and return to the Plate tab in Run Setup.

4. View the details of the plate and do one of the following:

n If the details are correct, click Next to proceed to the Start Run tab.
n If the details are incorrect, click Edit Selected to return to the Plate Editor. Revise the plate
file, save changes, and then click Next on the Plate tab to proceed to the Start Run tab.

To select an existing plate file

1. On the Plate tab, do one of the following:

n Click Select Existing and navigate to an existing plate file.

n Click Express Load and select a plate file from the dropdown list.

Tip: You can add plates to or remove them from the Express Load dropdown list. See
Adding and Removing Express Load Plate Files that follows for more information.

2. View the details of the plate and do one of the following:

n If the details are correct, click Next to proceed to the Start Run tab.
n If the details are incorrect, click Edit Selected to open the Plate Editor window. Revise the
plate file, save changes, and then click Next on the Plate tab to proceed to the Start Run tab.

Adding and Removing Express Load Plate Files

You can modify the contents of the Express Load dropdown list that appears in the Plate Editor.
The plates that appear in this list are saved in the following folder:

c:\Users\Public\Documents\Bio-Rad\CFX\Users\<user_name>\ExpressLoad\

To modify the Express Load list of plate files

1. Navigate to and open the ExpressLoad folder.

2. Review the plate files (.pltd) in the folder.

3. Do one of the following:

n Delete plate files from the folder to remove them from the dropdown list.

n Copy plate files into the folder to add them to the dropdown list.

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Chapter 7 Running Experiments

Start Run Tab

The Start Run tab displays information about the experiment to run. It also displays the connected
instrument block or blocks on which you can run the experiment.

In the Start Run tab you can do the following:

n View detailed run information, including the selected protocol file, plate file, and scan mode.

n Add notes about the run.

n View details about all connected instruments, including their run status (running or idle),
sample volume in µl, lid temperature, emulation mode, and ID or bar code if available.

Note: You can modify the columns that appear in the Start Run on Selected Blocks table.
See Modifying Details in the Selected Blocks Table on page 113 for information.

n Select the block or blocks on which to perform the run.

n Remotely open or close the lid of each selected instrument.

n Start the run.

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 Start Run Tab

Modifying Details in the Selected Blocks Table

You can modify the columns that appear in the Start Run on Selected Block(s) table. You can also
modify the default sample volume and lid temperature values in the table. The setting changes are
applied to the run to be performed.

To add columns in the Start Run on Selected Blocks table

u Right-click the table and select an option in the menu that appears.

To remove columns in the Start Run on Selected Blocks table

u Right-click the table and clear the option in the menu that appears.

To edit sample volume or lid temperature values for a block

u Select the sample volume or lid temperature cell for the target block and type a new value into
the cell.

To add a run ID or bar code for a block

u Select the ID/Bar Code cell for the target block and type an ID or scan the block with a bar
code reader.

Running an Experiment
To run an experiment

1. In the Start Run tab, verify the plate and protocol details in the Run Information section.

2. (Optional) Add notes about the run or experiment in the Notes text box.

3. Select the checkbox of one or more blocks on which to perform the run.

Tip: To run the experiment on all blocks, select Select All Blocks located below the
Selected Blocks table.

4. (Optional) Click Flash Block Indicator to flash the indicator LED on each selected instrument
block.

5. Insert experiment plates into the block:

a. Click Open Lid. The motorized lid of each selected block opens.

b. Insert an experiment block into each selected block.

c. Click Close Lid.

Tip: You can also press the button on the front of each block to open and close the lid.

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Chapter 7 Running Experiments

6. Click Open Lid and Close Lid to open and close the motorized lid of each selected instrument
block.

7. View the details of the run and do one of the following:

n If the details are correct, click Start Run.

n If the details are incorrect:

o Correct the details in the Selected Blocks table and click Start Run.

o Return to the correct tab and make the appropriate changes, save the changes and
then click Next to return to the Start Run tab and start the run.

To start a new run from a previous run

u Do one of the following:

n Select File > Repeat a Run in the main software menu bar; navigate to and double-click
the run data file that you want to repeat.

n Select the Repeat Run tab in the Startup Wizard and double-click the run data file of the
run you want to repeat.

Optionally, on the Repeat Run tab you can click Browse and navigate to and double-click
the run data file that you want to repeat.

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 Run Details Dialog Box

Run Details Dialog Box

When you click Start Run, CFX Maestro software prompts you to save the data file (.pcrd), starts the
run, and opens the Run Details dialog box. The Run Details dialog box comprises three status
tabs:

n Run Status — use this tab to view the current status of the protocol, open or close the lid,
pause a run, add repeats, skip steps, or stop the run.

n Real-time Status — use this tab to view the real-time PCR fluorescence data as they are
collected.

n Time Status — use this tab to view a full-screen countdown timer for the protocol.

These tabs are explained in detail in the sections that follow.

Run Status Tab

The Run Status tab displays the current status of a run in progress. In this view you can also control
the lid and change the run in progress.

LEGEND

1. Run Status pane — displays the current progress of the protocol.

2. Run Status controls — enable you to operate the instrument or to interrupt the
current protocol.

3. Run Information pane — displays run details.

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Chapter 7 Running Experiments

Run Status Commands

Use the commands in the Run Status tab to either operate the instrument from the software or
change a run that is in progress.

Note: Making changes to the protocol during the run, such as adding repeats, does not
change the protocol file associated with the run. These actions are recorded in the Run Log.

— opens the motorized lid on selected instruments.

Important: Opening the lid during a run pauses the run during the current step and might
alter the data.

— closes the motorized lid on selected instruments.

— adds more repeats to the current GOTO step in the protocol. This option is
available only when a GOTO step is running.

— skips the current step in the protocol.

Note: If you skip a GOTO step, the software prompts you to confirm that you want to skip
the entire GOTO loop and proceed to the next step in the protocol.

— flashes the LED on the selected instrument to identify the selected blocks.

— pauses the protocol.

Note: This action is recorded in the Run Log.

— resumes a paused protocol.

— stops the run before the protocols ends.

Note: Stopping a run before the protocol ends might alter your data.

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Real-time Status Tab

The Real-time Status tab displays real-time PCR data collected at each cycle during the run after
the first two plate reads.

LEGEND

1. Amplification trace pane — displays real-time amplification data during the run.

2. Well group identifier — if well groups were identified in the plate setup, users can
select a specific well group to view its traces, wells, and tabular information.
Step number identifier —if the protocol collects data at more than one step (for
example during amplification and melt curve), users can select a specific step and
view the traces collected at that step.

3. Well selector pane — displays the active, inactive, and empty wells in the plate.

4. Plate setup table pane — displays the plate setup in tabular format.

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5. Run details pane — displays the real-time status of the run including:
n Current step
n Current repeat
n Current temperature
n Time remaining
n Sample temperature
n Lid temperature

6. Plate Setup — opens the Plate Setup dialog box, in which users can modify the
current plate setup during a run.

In the Real-time Status tab you can:

n Show or hide real-time traces by selecting them in the well selector pane or the plate setup
table.

n View single or groups of traces by selecting them in the well-group dropdown.

n Edit the plate or replace the plate file.

n Apply a PrimePCR file to the run.

Showing or Hiding Real-Time Traces

By default, all filled wells are active and appear in the plate setup table. Active wells appear blue in
the well selector pane. Hidden wells appear light gray, and unused wells appear dark gray in the
well selector pane.

You can hide traces from active wells during the run. CFX Maestro continues to collect data for all
wells; when you hide wells, their data do not appear in the plate setup table.

To hide real-time traces

u In the well selector pane, click the active (blue) wells that you want to hide.

To show real-time traces

u In the well selector pane, click the hidden (light gray) wells that you want to display.

For more information about the well selector, see Well Selector on page 135.

Editing a Plate Setup

To edit a plate setup

u Click Plate Setup and then select View/Edit Plate.

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The Plate Editor window appears, in which you can edit the plate while the run is in progress.
For more information about editing plates, see Chapter 6, Preparing Plates.

Note: You can also edit the trace styles from the Plate Editor window. Changes appear in
the amplification trace plot in the Real-time Status tab.

Replacing a Plate File

Tip: Replacing a plate file is especially useful if you start a run with a Quick Plate file in the
ExpressLoad folder.

To replace a plate file

u Click Plate Setup and then select one of the following options:

n Replace Plate file — select the new plate file from the list in the browser window

n Apply PrimePCR file — search for a run file from which the plate layout will be obtained
using Smart search or click Browse to find a file that you downloaded from the Bio-Rad
website and that is not located in the PrimePCR folder

Note: CFX Maestro checks the scan mode and plate size for the plate file. These must be
the same as the run settings with which the run was started.

Time Status Tab

The Time Status tab displays the time remaining to complete the current run.

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Chapter 7 Running Experiments

Performing PrimePCR Experiments

PrimePCR experiments use pathway or disease-specific assays that Bio-Rad has wet-lab
validated and optimized and are available in the following formats:

n Preplated panels — plates containing assays that are specific for a biological pathway or
disease; they include PrimePCR controls and reference genes

n Custom configured plates — plates that can be set up in a user-defined layout with the option
to choose assays for targets of interest, controls, and references

n Individual assays — tubes that contain individual primer sets for use in real-time reactions

To reduce the overall run time, you can remove the melt step in the protocol. Bio-Rad strongly
recommends that you do not make any other modifications to a PrimePCR run protocol. The
default protocol is the one that was used for assay validation. Any deviation from this may affect the
results. Protocol changes are noted in the Run Information tab of the resultant data file and in any
reports that are created.

To start a PrimePCR run

u To start a PrimePCR run, do any of the following:

n In the Startup Wizard, select PrimePCR on the Run setup tab and then select the
appropriate chemistry (SYBER or probe).

n Select a PrimePCR run from the Recent Runs list on the Repeat run tab in the Startup
Wizard.

n Select File > New > PrimePCR Run on the Home window.

n Select File > Open > PrimePCR Run File on the Home window.

n Drag and drop a PrimePCR run file onto the Home window.

After you select a PrimePCR run, the Run Setup window opens on the Start Run tab with the
default PrimePCR plate layout loaded based on the selected instrument.

To remove the melt step in the protocol

u On the Protocol tab, clear the box adjacent to Include Melt Step.

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 Performing PrimePCR Experiments

To import target information for PrimePCR plates into a plate layout

1. Do one of the following:

n In the Real-time Status tab in the Run Details dialog box, select Plate Setup > Apply
PrimePCR File.

n In the Data Analysis window, select Plate Setup > Apply PrimePCR File.

2. In the PrimePCR run file dialog box, click Browse to navigate to the appropriate PrimePCR file
(.csv).

3. Select the target PrimePCR file and click Open.

CFX Maestro imports the target information into your plate layout.

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Chapter 7 Running Experiments

Transferring Stand-Alone Data for Analysis

When the run is complete, CFX Maestro analyses the fluorescence data. If the run is performed in
stand-alone mode and saved on the instrument itself, the data need to be transferred to the CFX
Maestro computer for analysis.

The instrument can store up to 100 real-time PCR runs. After the run completes, you can transfer
stand-alone data files to the CFX Maestro computer through email, USB drive, or through the
software itself.

This section explains how to transfer stand-alone data files to the CFX Maestro computer.

Transferring Data Through CFX Maestro Software

Important: When you transfer data files through CFX Maestro, all files saved on the instrument
are transferred. Ensure you have enough disk space for the data to transfer safely.

To transfer data from CFX Maestro

1. In the Detected Instruments pane on the Home window, right-click the target instrument and
select Retrieve Data Files.

CFX Maestro displays the Browse For Folder dialog box.

2. In the Browse For Folder dialog box, browse to the location in which you plan to save the data
files and click OK.

The transfer process creates a folder labeled Real-Time Data in the selected location. The run
data are saved into the Real-Time Data folder as separate .zpcr files.

Transferring Data Through Email

To email a data file at the end of a run

1. Set up email notifications for the instrument.

See Setting Up Email Notification on page 30 or the C1000 Touch Thermal Cycler and CFX
Real-Time PCR Detection Systems Instrument Guide.

2. When you set up email notifications, ensure that Attach Data File is selected.

The run data are emailed as a .pcrd file.

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Transferring Data Using a USB Drive

Tip: If you insert a USB drive into the USB port on the instrument, the data file is automatically
saved to the root directory of the USB drive when the run is complete. You can also locate
previously saved data files and save them onto an attached USB drive.

To locate and save data files onto a USB drive

1. On the instrument Home screen, touch Saved Files to access the file folders.

2. On the Location column, touch Real-Time Data.

3. Select the file to export in the File column.

Information about the selected file appears in the Preview pane.

4. To export the file, touch File Options.

5. Touch OK to save the file to the attached USB drive.

The run data are saved onto the USB drive as a .zpcr file.

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Chapter 7 Running Experiments

Creating a Data File

To analyze data transferred from the instrument to the CFX Maestro computer, the compressed
data file (.zpcr file) must be converted to a data file (.pcrd file). CFX Maestro converts the .zpcr file is
to a .pcrd file and then selects a plate file that has the same the scan mode and plate size and
applies it to the .pcrd file.

To create a data file from a stand-alone data file

1. In CFX Maestro do one of the following:

n Locate the target .zpcr file and drag it onto the CFX Maestro Home window.

n Select File > Open > Stand-alone Run and navigate to and select the target file.

CFX Maestro displays the Save As dialog box.

2. Navigate to the folder in which you plan to save the .pcrd file and click Save.

After you save the .pcrd file, CFX Maestro opens the Data Analysis window and displays the
resulting data.

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Chapter 8 Data Analysis Overview
CFX Maestro™ software processes real-time PCR data automatically at the end of each run and
opens the Data Analysis window to display these data (the .pcrd file).

CFX Maestro software offers several methods to open and view data files. You can:

n Drag a data file (.pcrd extension) onto the Home window and release it.

n Select File > Open > Data File in the Home window and browse to the target .pcrd file.

n Select File > Recent Data Files in the Home window to select from a list of the ten most
recently opened data files.

n Choose the Analyze tab in the Startup Wizard and either select from Recent Files or click
Browse to locate the data file.

Data Analysis Window

The Data Analysis window displays multiple tabs, each tab showing the analyzed data for a
specific analysis method or run-specific information. Tabs appear only if the data collected in the
run are available for that type of analysis.

Tip: To choose the tabs to display, select them from the dropdown menu View in the Data
Analysis window. To return to the original tab layout, select Settings > Restore Default Window
Layout.

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Data Analysis Toolbar

The toolbar in the Data Analysis window provides quick access to important data analysis
functions.

Table 10 lists the functions of buttons in the toolbar.

Table 10. Toolbar in the Data Analysis window

Button Name Function

Plate Setup View/Edit plate: Opens the Plate Editor to view
and edit the contents of the wells.
Replace Plate file: Selects a plate file to replace
the plate layout.
Apply PrimePCR file: Selects a run file to replace
the plate layout for a PrimePCR™ run.

Manage Well Opens the Well Groups Manager window to

Groups create, edit, and delete well groups.

Well Group Selects an existing well group name from the

dropdown menu. The default selection is All
Wells. This button appears only when well groups
are created.

Analysis Mode Analyzes the data in either Fluorophore or Target

mode.

Help Opens the software Help for more information

about data analysis.

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Data Analysis Menu Bar

Table 11 lists the menu bar items in the Data Analysis window.

Table 11. Data Analysis window menu bar items

Menu Item Command Function

File Save Saves the file.

Save As Saves the file with a new name.

Repeat Run Extracts the protocol and plate file from the
current run to rerun it.

Close Closes the Data Analysis window.

View Run Log Opens a Run Log window to view the run log
of the current data file.

Quantification, Melt Curve, Gene Displays the analyzed data in selected tabs
Expression, End Point, Custom in the Data Analysis window. At least one tab
Data View, QC, Run Information must be selected.

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Table 11. Data Analysis window menu bar items, continued

Menu Item Command Function

Settings Cq Determination Mode Select Regression or Single Threshold mode
to determine how Cq values are calculated
for each trace.

Baseline Setting Select Baseline Subtraction method for the

selected well groups.

Analysis Mode Select to analyze data by Fluorophore or by

Target.

Cycles to Analyze Select the cycles that are to be analyzed.

Baseline Thresholds Opens the Baseline Thresholds window to

adjust the baseline or the threshold.

Trace Styles Opens the Trace Styles window.

Plate Setup Opens the Plate Editor to view and edit the
plate; replace the current plate with one from
a user-defined plate file or a PrimePCR run
file.

Include All Excluded Wells Includes all excluded wells in the analysis.

Mouse Highlighting Turns on or off the simultaneous highlighting

of data with the mouse pointer.
Tip: If Mouse Highlighting is turned off,
press the Control key to temporarily turn
on highlighting.

Restore Default Window Layout Restores the arrangement of windows to the

default setting.

Export Export All Data Sheets to Excel Exports all the spreadsheet views from every
tab to a separate Excel file.

Export RDML File Select a version of RDML (1.1 or 1.0) to open

a Save As window to specify an RDML file
name and location.

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Table 11. Data Analysis window menu bar items, continued

Menu Item Command Function

Custom Export Opens the Custom Export window in which
the fields to be exported and the file format
can be specified.

Export to LIMS Folder Opens a window to save data in a

predetermined format to the LIMS folder.

Tools Reports Opens the Report for this data file.

Well Group Reports Opens the Well Group Report window to

generate reports for specified well groups.

Import Fluorophore Calibration Select a calibration file to apply to the current

data file.

qbase+ Launches qbase+ v2.5 directly from current

.pcrd file if it is installed.

Tab Details
Each tab in the Data Analysis window displays data in charts and spreadsheets for a specific
analysis method and includes a well selector to select the data you want to show. When it opens,
the Data Analysis displays the Quantification tab by default. You can use the Amplification chart
data in the Quantification tab to determine the appropriate analysis settings for the run.

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Chapter 8 Data Analysis Overview

Note: The software links the data in the panes of each Data Analysis tab. For example,
highlighting a well by placing the mouse pointer over the well in the well selector view
highlights the data in all the other panes.

Step Number Selector

The CFX96 Touch™, CFX96 Touch Deep Well, CFX Connect™, and CFX384 Touch™ systems
can acquire fluorescence data at multiple protocol steps; the software maintains the data acquired
at each step independently. The software displays the Step Number selector below the Standard
Curve chart on the Quantification tab. When a protocol contains at least one data collection step,
CFX Maestro software displays the data from the first collection step.

If the protocol contains more than one collection step, you can select another the step from the
dropdown list, for example:

When you select a step, the software applies that selection to all the data that are shown in the
Data Analysis window.

Viewing Well Groups in Data Analysis

Wells in the plate can be grouped into subsets for independent analysis using well groups. When
you create well groups, their group names appear in the Data Analysis window Well Groups
dropdown list on the toolbar.

If you created well groups, the software displays the default well group All Wells when you open
the Data Analysis window, displaying the data in all wells with content in the charts and
spreadsheets. Only the wells in that well group loaded with content appear in the well selector, and
only data for those wells are included in the data analysis calculations.

Tip: To create, edit, and delete well groups, click Manage Well Groups in the toolbar.

Note: If you did not create well groups, the Well Groups dropdown list does not appear in the
toolbar.

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Changing Well Contents after a Run

During data analysis, changing the way the data are displayed by changing the contents of the
wells in the Plate Editor never changes the fluorescence data that were collected from each well
during the run. After the module collects fluorescence data, you cannot delete those data but you
can choose to remove data from view and analysis.

To change the content of wells after a run

u In the Data Analysis window, click Plate Setup and select one of the following options:

n Edit/View Plate — opens the Plate Editor, in which you can make manual changes to the
layout.

n Replace Plate file —opens the Select Plate browser, in which you can navigate to a
previously saved plate file with which to replace the current plate layout.

n Apply PrimePCR file — opens the Select PrimePCR file dialog box, in which you can
navigate to a PrimePCR™ run file and apply it to the plate layout.

Tip: You can add or edit information about the contents of the well before a run, during a run,
or after a PCR run completes. You must assign the scan mode and plate size before the run.
These parameters cannot change after the run.

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Data Analysis Settings

The Amplification chart data in the Quantification tab show the relative fluorescence (RFU) for each
well at every cycle. Each trace in the chart represents data from a single fluorophore in one well.
These data are used to determine Cq values for each well on a per fluorophore basis. The software
uses one of two modes to determine Cq values:

n Regression — applies a multivariable, nonlinear regression model to individual well traces

and then uses this model to compute an optimal Cq value.

n Single Threshold — uses a single threshold value to calculate the Cq value based on the
threshold crossing point of individual fluorescence traces.

Select Settings > Cq Determination Mode to choose the Cq determination mode.

Adjusting the Threshold

In Single Threshold mode, you can adjust the threshold for a fluorophore by clicking on the
threshold line in the Amplification chart and moving the mouse pointer vertically. Alternatively, you
can specify an exact crossing threshold for the selected fluorophore.

Baseline Settings
The software automatically sets the baseline individually for each well. The baseline setting
determines the method of baseline subtraction for all fluorescence traces. The software provides
three baseline subtraction options:

n No Baseline Subtraction — displays the data as relative fluorescence traces. Some analysis
is not possible in this analysis mode and therefore the software does not display the Gene
Expression, End Point, and Allelic Discrimination tabs.

n Baseline Subtracted — displays the data as baseline subtracted traces for each fluorophore
in a well. The software must baseline subtract the data to determine quantification cycles,
construct standard curves, and determine the concentration of unknown samples. To generate
a baseline subtracted trace, the software fits the best straight line through the recorded
fluorescence of each well during the baseline cycles and then subtracts the best fit data from
the background subtracted data at each cycle.

n Baseline Subtracted Curve Fit — displays the data as baseline subtracted traces and the
software smooths the baseline subtracted curve using a centered mean filter. This process is
performed so that each Cq is left invariant.

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 Data Analysis Settings

In addition to these options, you can also select Apply Fluorescent Drift Correction. For wells that have
abnormally drifting RFU values during the initial few cycles of a run, the software derives an estimated
baseline from adjacent wells for which a horizontal baseline was successfully generated.

To change the baseline subtraction setting

u Select Settings > Baseline Setting.

Analysis Mode
Data can be grouped and analyzed by either fluorophore or target name. When grouped by
fluorophore, data traces are displayed by fluorophore as indicated in the plate setup for that run.
Individual fluorophore data appear in the amplification and standard curve chart (if available) when
the appropriate fluorophore selector checkboxes, located below the amplification chart, are selected.

When grouped by target, data traces are displayed by target name as entered in the plate setup for
that run.

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Chapter 8 Data Analysis Overview

To choose a data analysis mode

u Do one of the following:

n Select Settings > Analysis Mode.

n Choose a mode from the Analysis Mode dropdown menu in the toolbar.

Cycles to Analyze
You can restrict the number of cycles to analyze. You can also analyze data from a specific set of
cycles. The maximum number cycles you can analyze is 50.

Note: Removing cycles from the beginning of a run can have a significant impact on
baselining.

To restrict data analysis to a specific range of cycles

1. Select Settings > Cycles to Analyze.

The Cycles to Analyze dialog box appears.

2. Enter the starting and ending cycle values and click OK.

Click Restore Defaults in the Cycles to Analyze dialog box to return to the cycles originally used for
analysis.

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Well Selector
Use the Well Selector to display or hide the well data in the charts or spreadsheets throughout the
Data Analysis window. Only wells loaded with sample can be selected in the well selector. The
software colors the wells in the Well Selector:

n Blue — indicates selected wells. The data from selected wells appear in the Data Analysis
window.

n Light gray — indicates unselected wells. The data from unselected wells do not appear in the
Data Analysis window.

n Dark gray — indicates empty wells.

To display or hide well data

u In the well selector, do any of the following:

n To hide one well, click the individual well. To display that well, click the well again.

n To hide multiple wells, drag across the wells you want to select. To display those wells,
drag across the wells again.

n Click the top left corner of the plate to hide all the wells. Click the top left corner again to
display all wells.

n Click the start of a column or row to hide those wells. Click the column or row again to
display the wells.

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Chapter 8 Data Analysis Overview

Well Selector Right-Click Menu Items

Table 12 lists the right-click options available in the well selector view.

Table 12. Right-click menu items in the well selectors

Item Function
Well XX Displays only this well, removes this well from view, set color for
this well, or excludes this well from analysis.

Selected Wells (right-click and Displays only these wells, removes these wells from view, sets
drag) color for these wells, or excludes these wells from analysis.

Copy Copies the content of the well to a clipboard, including Sample Type
and optional Replicate #.

Copy as Image Copies the well selector view as an image.

Print Prints the well selector view.

Print Selection Prints the current selection.

Export to Excel Exports the data to an Excel spreadsheet.

Export to Csv Exports the data as a text document.

Export to Xml Exports the data as an .xml document.

Well Labels Changes the well labels to Sample Type, Target Name, or Sample
Name.

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 Well Selector

Temporarily Excluding Wells from Analysis

To exclude wells from data analysis temporarily

1. Right-click the well in the well selector, on a fluorescence trace, or on a point plotted on the
standard curve. To exclude multiple wells, right-click and drag to highlight multiple wells,
traces, or points.

2. From the right-click menu, choose the appropriate option:

n Well > Exclude Well

n Selected Wells > Exclude from Analysis

n Selected Traces > Exclude these wells from Analysis

Alternatively, to permanently remove wells from analysis, clear the contents from wells in the
Plate Editor by clicking the Clear Wells button.

Important: You must reenter any well content that is cleared.

To include an excluded well

u Right-click the appropriate well in the well selector and select Well > Include Well in Analysis.

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Chapter 8 Data Analysis Overview

Charts
Each chart in the Data Analysis window displays the data in a different graph and includes options
for adjusting the data.

Chart Tools
Chart tools appear in each chart in the Data Analysis window. All charts display the following tools:

Copy to Clipboard — copies the contents of the chart view to the clipboard.

Chart Settings — opens the Chart Settings dialog box in which you can modify the chart’s display
options including:

n Chart and axis titles

n Chart and axis font and size

n Axis scale

n Legend position

Export — opens the Export Options dialog box, from which you can modify the resolution and size
of the graph and save it to a specified location as one of the following file types:

n .bmp

n .jpg

n .png

Bar Chart Tools

In addition to the chart tools, bar charts display the following tools:

Sort — sorts the targets and samples alphabetically or in reverse alpha order.

Color Settings — opens the Color Settings dialog box, in which you can change the color of the
targets and samples.

For more information about these tools, see Changing and Annotating the Chart View on page
195.

Amplification Chart Tools

In addition to those listed above, amplification charts display the following tools:

Trace Styles — opens the Trace Styles dialog box, in which you can modify the appearance of
traces in the amplification chart.

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 Charts

Baseline Threshold — opens the Baseline Threshold dialog box, in which you can modify the
default baseline for selected wells or change the threshold for each fluorescence curve in the
amplification chart.

Copying Chart Data to the Clipboard

You can copy the contents of the chart view and paste it into any application that accepts bitmap
image files.

To copy chart data to the clipboard

1. From the chart tools, select Copy to Clipboard.

2. Open an application that accepts bitmap images, for example Microsoft Word.

3. Right-click and select Paste to paste the bitmap image from the clipboard into the application.

Changing the Chart Display Settings

Use the Chart Settings dialog box to change the titles, fonts and sizes, axis scale, and legend
location for the displayed chart. Changes that you make apply to the displayed chart only and are
saved with the chart.

To change chart display settings

1. From the chart tools, click Chart Settings.

The Chart Settings dialog box appears.

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Chapter 8 Data Analysis Overview

2. Select the Chart And Axes tab to:

n Type a title for the chart.

n Type a new title for the x-axis and angle the labels.

n Type a new title for the y-axis.

3. Select the Fonts tab to change the chart’s font and font size.

Tip: By default, the font size autoscales as the chart size changes. Select Change Font
Size to set a static font size for each label type.

4. Select the Axes Scale tab to:

n Clear x- and y-axis autoscaling and specify minimum and maximum scaling values.

n Choose to display grid lines or tick marks on the graph.

5. Select the Legend tab to:

n Choose to hide the chart legend.

n Change the default position of the chart legend.

Note: When the legend is positioned to the left or right of the chart, it displays only the first
ten fluorophores in the chart.

6. Click Apply at any time to view chart setting changes without saving the changes.

7. Click OK to save the changes and return to the chart.

Exporting the Chart

Use this dialog box to modify the width, height, and resolution of the graph to export it in one of the
following file formats:

n .bmp

n .jpg

n .png

You can the use the exported graph to display your results in poster sessions, Microsoft
PowerPoint presentations, and professional journals.

Note: Consider the following when modifying the settings:

n Maximum and minimum width and height limits

o At 72 dpi: 0.1–83 in

o At 96 dpi: 0.1–62 in

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 Charts

o At 150 dpi: 0.1–40 in

o At 300 dpi: 0.1–20 in

o At 600 dpi: 0.1–10 in

o At all resolutions: 2–6,000 pixels

n Aspect ratio is based on width.

To export the chart

1. From the chart tools, click Export.

The Export Preview dialog box appears.

2. Modify the settings for the display as required.

3. Click Export.

4. In the Export dialog box, do the following:

a. (Optional) Navigate to a folder in which to save the chart file.

b. Type a name for the file and choose a file type from the dropdown list.

5. Click Save to save the chart file.

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Modifying the Baseline Threshold Settings

In Single Threshold mode, you can adjust the threshold for a fluorophore by clicking on the
threshold line in the Amplification chart and moving the mouse pointer vertically. Alternatively, you
can specify an exact crossing threshold for the selected fluorophore.

Tip: You can specify a cycle range to determine the baseline for all data files in the Data
Analysis tab in User > User Preferences.

To adjust the begin and end baseline cycle for each well

1. In the Quantification tab, select a single fluorophore under the Amplification chart.

2. From the chart tools, select Baseline Threshold.

The Baseline Threshold dialog box appears.

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 Charts

3. In the Baseline Cycles section, do one of the following:

n To select one well, click its row number.

n To select multiple adjacent wells, click the row number of the first well and drag down the
column to the final well.

n To select multiple nonadjacent wells, press the Control key and click the row number of
each target well.

n To select all wells, click the top left corner on the table.

4. Adjust the Baseline Begin cycle and Baseline End cycle for all selected wells, or change the
Begin and End cycle number at the bottom of the spreadsheet.

Tip: To revert the settings back to the last saved values, click Reset All User Defined
Values.

5. Click OK to save the changes and return to the chart.

To specify a cycle range for all data files

u In the Home or Plate Editor window, select User > User Preferences and choose the Data
Analysis tab.

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Chapter 8 Data Analysis Overview

Sorting Target, Sample, and Biological Group Data

Note: This option is available on gene expression charts only.

By default, the Targets, Samples, and Biological Groups lists appear in alphabetical order. Use the
Sort dialog box to sort the display in reverse alpha order or to manually move a term to a different
position in the list.

To sort target, sample, and biological group data

1. From the chart tools, click Sort.

The Gene Expression Chart Sorting dialog box appears.

2. In the dialog box, click Z-A to sort the list in reverse alphabetical order.

3. To manually move a term, select it and click the appropriate button between the charts:

n Click the Up or Down arrow to move the selected term one position.

n Click the Up or Down bar arrow to move the selected term to the top or bottom of the list.

4. Click OK to save the changes and return to the Gene Expression tab.

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 Charts

Changing the Target and Sample Color Settings

Note: This option is available on gene expression charts only.

Use the Color Settings dialog box to change the color of a target or sample, or to remove the item
from the graph.

To change color settings

1. From the chart tools, select Color Settings.

The Color Settings dialog box appears.

2. To change the display color for a target or sample, click its color in the Color column.

3. In the Color dialog box that appears, select a new color and click OK.

4. To remove the item from the gene expression graph, clear its checkbox in the Show Chart
column.

Tip: To clear all items from the gene expression graph, clear the Show Chart checkbox in
the column head.

5. (Optional) By default, the bar chart color appears in gradient form. To display the color in solid
form, select Use Solid Colors.

6. Click OK to save the changes and return to the Gene Expression tab.

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Chapter 8 Data Analysis Overview

Magnifying an Area in the Chart

To magnify an area of the chart

u Click and drag across the chart. The software resizes the chart and centers it on the selected
area.

To reset the chart to full view

u Right-click in the chart and select Set Scale to Default.

Copying Charts into a Microsoft File

You can copy data charts into Microsoft Word, Excel, or PowerPoint documents. The image
resolution corresponds to that of the screen from which the image was obtained

To copy charts into a Microsoft file

1. In the Data Analysis window, click Copy To Clipboard in the upper right corner of the chart’s
pane.

2. Open a blank Microsoft file and paste the contents from the clipboard.

Common Right-Click Menu Items for Charts

Table 13 lists the right-click menu items that are available on charts. Some of the available items
are present for all charts, and these items can be used to change how the data are displayed or to
easily export the data from a chart.

Table 13. Right-click menu items for charts

Item Function
Copy Copies the chart into the clipboard.

Save Image As Saves the image at a specified size, resolution, and file type. The image
formats available are PNG (default), JPG, and BMP.

Set Scale to Default Returns the chart to its default view after magnifying the chart.

Chart Options Opens the Chart Options window to change the chart, including
changing the title, selecting limits for the x and y axes, showing grid
lines, and showing minor ticks in the axes.

Note: Menu items that apply to specific charts are described in Chapter 9, Data Analysis
Details.

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 Spreadsheets

Spreadsheets
The spreadsheets shown in Data Analysis include options for sorting and transferring the data.
Sort the columns by one of these methods:

n Click and drag a column to a new location in the selected table.

n Click the column header to sort the data in ascending or descending order.

To sort up to three columns of data in the Sort window

1. Right-click in the spreadsheet and select Sort.

2. In the Sort dialog box, select the first column title to sort. Sort the data in ascending or
descending order.

3. Select a second or third column to sort and choose Ascending or Descending.

4. Click OK to sort the data or click Cancel to stop sorting.

Highlight the data on the associated charts and well selector by holding the mouse pointer over a
cell. Click in a cell to copy and paste its contents into another software program.

Common Right-Click Menu Items for Spreadsheets

Table 14 lists the right-click menu items available on any spreadsheet view.

Table 14. Right-click menu items for spreadsheets

Item Function
Copy Copies the contents of the selected wells to a clipboard, then paste
the contents into a spreadsheet such as Excel.

Copy as Image Copies the spreadsheet view as an image file and paste it into a file
that accepts an image file, such as text, image, or spreadsheet
files.

Print Prints the current view.

Print Selection Prints the current selection.

Export to Excel Exports the data to an Excel spreadsheet.

Export to Text Exports the data to a text editor.

Export to Xml Exports the data to an Xml file.

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Chapter 8 Data Analysis Overview

Table 14. Right-click menu items for spreadsheets, continued

Item Function
Export to Html Exports the data to an Html file.

Find Searches for text.

Sort Sorts the data in up to three columns.

Select Columns Selects the columns that will be displayed in the spreadsheet.

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 Export

Export
CFX Maestro software provides four export options from the Export dropdown menu:

n Export All Data Sheets to Excel

n Export RDML Files

n Custom Export

n Export to LIMS

Exporting All Data Sheets to Excel

You can export all the spreadsheet views from every tab of CFX Maestro software into individual
Excel files.

To export all data sheets to Excel

u Select Export > Export All Data Sheets to Excel.

Exporting RDML Files

RDML is a structured and universal data standard for exchanging quantitative PCR (qPCR) data.
The data standard is a text file in Extensible Markup Language (.xml) format. Refer to the
International RDML Consortium website (www.rdml.org) for additional information about the RDML
data exchange format.

Note: Save the RDML file as version 1.1 if you are using version 2.3 or higher of qbase+
software.

To export an RDML file

1. Select Export > Export RDML Files and select RDML v1.1 or RDML v1.0 from the list that
appears.

The Save As dialog box appears.

2. In the Save As dialog box, specify a file name and location in which to save the RDML file.

3. Click OK to save the export file.

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Chapter 8 Data Analysis Overview

Creating a Custom Export File

To create a custom export file

1. Select Export > Custom Export. The Custom Export dialog box appears.

2. Select the export format from the dropdown list that appears.

3. Select the checkboxes for the items to export.

4. (Optional) Click Customize Column Names to change column names.

5. Click Export. The Save As dialog box appears.

6. In the Save As dialog box, specify a file name and location in which to save the exported file.

7. Click OK to save the export file.

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 Export

Exporting to a LIMS Folder

You can export data into a LIMS-compatible file format. For more information about creating,
managing, and using LIMS files, see Appendix C, LIMS Integration.

To export data in LIMS format

1. Select Export > Export to LIMS Folder.

The Save As dialog box appears.

2. In the Save As dialog box, specify a file name and location in which to save the exported file.

3. Click OK to save the export file.

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Chapter 8 Data Analysis Overview

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Chapter 9 Data Analysis Details
The Data Analysis window comprises multiple tabs from which to view data. This chapter explains
these tabs in detail.

Tip: You can choose which tabs to view in the Data Analysis window using the View menu.
The customized layout is saved with the data file.

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Chapter 9 Data Analysis Details

Quantification Tab
Use the data in the Quantification tab to set the data analysis conditions, including the baseline
settings for individual wells and the threshold settings. The Quantification tab displays data in
these four views:

n Amplification chart — displays the relative fluorescence units (RFU) for each well at every
cycle. Each trace in the chart represents data from a single fluorophore in one well.

n Standard curve — appears only if the run includes wells designated as sample type standard
(Std). The standard curve displays the threshold cycle plotted against the log of the starting
quantity. The legend displays the Reaction Efficiency (E) for each fluorophore in the wells with
a Standard sample type.

n Well selector — selects the wells with the fluorescence data you want to show.

n Spreadsheet — displays a spreadsheet of the data collected in the selected wells.

Fluorophore Options
To display flourophore data in the Quantification tab charts and spreadsheets, select the target
fluorophore(s) below the Amplification chart. To hide the fluorophore data in the data analysis
window, clear its checkbox.

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 Quantification Tab

Trace Styles Dialog Box

Using the Trace Styles dialog box, you can adjust the appearance of traces in the amplification and
melt curve charts in the Quantification and Melt Curve tabs. You can then preview the changes in
the well selector that appears in the Trace Styles dialog box.

To adjust trace styles

1. Select only one fluorophore under the Amplification chart.

2. To open the Trace Styles dialog box, do one of the following:

n Click Trace Styles in the Amplification chart.

n Select Settings > Trace Styles in the Data Analysis menu bar.

n Right-click on a trace and select Trace Styles.

The Trace Styles dialog appears.

3. In the Trace Styles dialog box, select a specific set of wells in the well selector in the bottom
pane. Alternatively, select wells that contain one sample type in the dropdown menu in the
Wells column.

4. Do any of the following:

n To choose a color for the selected wells, click the box in the Color column.

n To assign a symbol to the selected wells, select a symbol from the Symbol dropdown list.

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Chapter 9 Data Analysis Details

n To quickly color the wells by button label, click the appropriate quick set:

o Random by Well

o Random by Replicate

o Use Fluor Colors

o Use Target Colors

o Use Sample Colors

n To assign well labels, choose either Sample Type, Target Name, Sample Name, or
Symbol.

Log Scale Option

Select Log Scale below the Amplification chart to view the fluorescence traces in a semilog scale:

Tip: To magnify any area of the chart, drag across the target area. To return to a full view, right-
click on the chart and select Set Scale to Default.

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 Quantification Tab

Standard Curve Chart

The software creates a Standard Curve chart in the Quantification tab if the data include sample
types defined as Std for at least one fluorophore in the run.

The Standard Curve chart displays the following information:

n Name for each curve (the fluorophore or target).

n Color of each fluorophore or target.

n Reaction efficiency (E). Use this statistic to optimize a multiplex reaction and to equalize the
data for a standard curve.

Note: The reaction efficiency describes how much of your target is being produced with
each cycle in the protocol. An efficiency of 100% indicates that you are doubling your
target with each cycle.

n Coefficient of determination, R2 (written as R^2). Use this statistic to determine how correctly
the line describes the data (goodness of fit).

n Slope

n y-intercept

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Chapter 9 Data Analysis Details

Amplification Chart Menu Options

In addition to the common right-click menu options to copy, print, and export charts, Table 15 lists
the menu options available only on the Amplification chart.

Table 15. Amplification chart right- and left-click menu items

Menu Option Function

Well XX, Fluor Target Displays only this well, removes this well from view, sets color for
this trace, or excludes this well from analysis.

Selected Traces Displays only these wells, removes these wells from view, sets
color for these traces, or excludes these wells from analysis.

Show Threshold Values Displays the threshold value for each amplification curve on the
chart.

Trace Styles Opens the Trace Styles window to change trace styles that appear
on the Quantification and Melt Curve tabs.

Baseline Thresholds Opens the Baseline Thresholds window to change the baseline or
thresholds of each fluorophore (changes appear in the
Amplification chart in the Quantification tab).

Quantification Tab Spreadsheet

Table 16 defines the data displayed in the spreadsheet in the Quantification tab.

Table 16. Quantification tab spreadsheet content

Information Description
Well Well position in the plate

Fluor Fluorophore detected

Target Target Name loaded in the Plate Editor wells

Content A combination of the Sample Type (required) and Replicate #

(optional) loaded in the Plate Editor

Sample Sample Name loaded in the Plate Editor wells

Cq Quantification cycle for each trace

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 Quantification Tab

Changing Target, Content, or Sample Data

You can change the data in the Target, Content, and Sample columns by editing the plate file
using the Plate Editor even after you run the experiment.

To change the data in the Content, Target, and Sample columns

u Click Plate Setup and select View/Edit Plate to open the Plate Editor.

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Chapter 9 Data Analysis Details

Quantification Data Tab

The Quantification Data tab displays the quantification data collected in each well. CFX Maestro
software displays the data in four different spreadsheet views:

n Results — displays a spreadsheet of the data. This is the default view.

n Standard Curve Results — displays a spreadsheet of the standard curve data.

n Plate — displays the data in each well as a plate map.

n RFU — displays the RFU quantities in each well for each cycle.

Select each spreadsheet from the dropdown list that appears below the Quantification Data tab.

Results Spreadsheet
The Results spreadsheet displays data for each well in the plate.

Note: All Std. Dev (standard deviation) calculations apply to the replicate groups assigned in
the wells in the Plate Editor window. The calculations average the Cq value for each well in
the replicate group.

Table 17 defines the data that appear in the Results spreadsheet.

Table 17. Results spreadsheet content

Information Description
Well Well position in the plate

Fluor Fluorophore detected

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 Quantification Data Tab

Table 17. Results spreadsheet content, continued

Information Description
Target Amplification target name (gene)

Content Sample type and Replicate #

Sample Sample description

Biological Set Name Name of the biological set

Cq Quantification cycle

Cq Mean Mean of the quantification cycle for the replicate group

Cq Std. Dev Standard deviation of the quantification cycle for the replicate group

Starting Quantity (SQ) Estimate of the starting quantity of the target

Log Starting Quantity Log of the starting quantity

SQ Mean Mean of the starting quantity

SQ Std. Dev Standard deviation of the starting quantity across replicates

Set Point Temperature of sample in the well for a gradient step across
replicates

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Chapter 9 Data Analysis Details

Standard Curve Results Spreadsheet

The Standard Curve Results spreadsheet displays the calculated standard curve parameters.

Table 18 defines the data that appear in the Standard Curve Results spreadsheet.

Table 18. Standard Curve Results spreadsheet contents

Information Description
Fluor (or Target) Fluorophore (or Target) detected

Efficiency % Reaction efficiency

Slope Slope of the standard curve

Y-intercept Point at which the curve intercepts the y-axis

R^2 Coefficient of determination

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 Quantification Data Tab

Plate Spreadsheet
The Plate spreadsheet displays a plate map of the data for one fluorophore at a time.

To view data for a specific fluorophore

u Click its tab at the bottom of the spreadsheet.

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Chapter 9 Data Analysis Details

RFU Spreadsheet
The RFU spreadsheet displays the relative fluorescence units (RFU) readings for each well
acquired at each cycle of the run. The well number appears at the top of each column and the
cycle number appears to the left of each row.

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 Melt Curve Tab

Melt Curve Tab

For DNA-binding dyes and noncleavable hybridization probes, the fluorescence is brightest when
the two strands of DNA anneal. Therefore, as the temperature rises towards the melting
temperature (Tm), fluorescence decreases at a constant rate (constant slope). At the Tm there is a
dramatic reduction in the fluorescence with a noticeable change in slope. The rate of this change is
determined by plotting the negative first regression of fluorescence versus temperature (-d
(RFU)/dT). The greatest rate of change in fluorescence results in visible peaks and represents the
Tm of the double-stranded DNA complexes.

The software plots the RFU data collected during a melt curve as a function of temperature. To
analyze melt peak data, the software assigns a beginning and ending temperature to each peak by
moving the threshold bar. The floor of the peak area is specified by the position of the melt
threshold bar. A valid peak must have a minimum height relative to the distance between the
threshold bar and the height of the highest peak.

The Melt Curve tab displays the Tm of amplified PCR products in four views:

n Melt Curve — displays the real-time data for each fluorophore as RFUs per temperature for
each well.

n Melt Peak — displays the negative regression of the RFU data per temperature for each well.

n Well selector — displays wells to show or hide the data.

n Peak spreadsheet — displays the data collected in the selected well.

Note: This spreadsheet displays up to two peaks for each trace. To see more peaks, click
the Melt Curve Data tab.

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Chapter 9 Data Analysis Details

Adjusting Melt Curve Data

To adjust the Melt Curve data

u Do any of the following:

n Click and drag the threshold bars in the Melt Peak chart to include or exclude peaks in
data analysis.

n Select Positive in the Peaks dropdown menu to show the spreadsheet data for the peaks
above the Melt Threshold line or select Negative to view the spreadsheet data for the
peaks below the Melt Threshold line.

n Open the Trace Styles window to change the color of the traces in the Melt Curve and Melt
Peak charts.

n Select a number in the Step Number selector to view the Melt Curve data at another step
in the protocol. The list shows more than one step if the protocol includes plate reads in
more than one melt curve step.

n Select wells in the well selector to focus on subsets of the data.

n Select a well group to view and analyze a subset of the wells in the plate. Select each well
group by name in the Well Group dropdown menu in the toolbar.

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 Melt Curve Data Tab

Melt Curve Data Tab

The Melt Curve Data tab displays the data from the Melt Curve tab in multiple spreadsheets that
include all the melt peaks for each trace. CFX Maestro software offers four spreadsheet options in
which to view the melt curve data:

n Melt Peaks — displays all the data, including all the melt peaks, for each trace. This is the
default view.

n Plate — displays a view of the data and contents of each well in the plate.

n RFU — displays the RFU quantities at each temperature for each well.

n -d(RFU)/dT — displays the negative rate of change in RFU as the temperature (T) changes.
This is a first regression plot for each well in the plate.

Select each spreadsheet from the dropdown list that appears below the Melt Curve Data tab.

Melt Peaks Spreadsheet

The Melt Peaks spreadsheet displays all melt curve data.

Table 19 on page 168 defines the data that appear in the Melt Peaks spreadsheet.

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Chapter 9 Data Analysis Details

Table 19. Melt Peaks spreadsheet content

Information Description
Well Well position in the plate

Fluor Fluorophore detected

Content Sample Type listed in the Plate Editor window

Target Amplification target (gene)

Sample Sample Name listed in the Plate Editor window

Melt Temperature The melting temperature of each product, listed as one peak
(highest) per row in the spreadsheet

Peak Height Height of the peak

Begin Temperature Temperature at the beginning of the peak

End Temperature Temperature at the end of the peak

Plate Spreadsheet
The Plate spreadsheet displays melt curve data in a plate format.

Note: To adjust the peak that the software calls, adjust the threshold line in the Melt Peak chart
on the Melt Curve tab.

Table 20 on page 169 defines the data that appear in the Plate spreadsheet.

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 Melt Curve Data Tab

Table 20. Plate spreadsheet content

Information Description
Content A combination of Sample Type (required) and Replicate # (optional)

Sample Sample description

Peak 1 First melt peak (highest)

Peak 2 Second (lower) melt peak

RFU Spreadsheet
The RFU spreadsheet displays the fluorescence for each well at each cycle acquired during the
melt curve.

Table 21 defines the data displayed in the RFU spreadsheet.

Table 21. RFU spreadsheet content

Information Description
Well number (A1, A2, A3, A4, A5) Well position in the plate for the loaded wells

Temperature Melting temperature of the amplified target,

plotted as one well per row and multiple wells
for multiple products in the same well

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Chapter 9 Data Analysis Details

-d(RFU)/dT Spreadsheet
The -d(RFU)/dT spreadsheet displays the negative rate of change in RFU as the temperature (T)
changes.

Table 22 defines the data that appear in the -d(RFU)/dT spreadsheet.

Table 22. -d(RFU)/dT spreadsheet content

Information Description
Well number (A1, A2, A3, A4, A5) Well position in the plate for the loaded wells

Temperature -d(RFU)/dT Negative rate of change in RFU as temperature (T)

changes

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 End Point Tab

End Point Tab

Open the End Point tab to analyze final relative fluorescence units (RFUs) for the sample wells.
The software compares the RFU levels for wells with unknown samples to the RFU levels for wells
with negative controls and “calls” the unknown positive or negative. Positive samples have an RFU
value that is greater than the average RFU value of the negative controls plus the cut off value.

To analyze the end-point data, the plate must contain negative controls or the software cannot
make the call. Run one of these two types of protocols:

n Run a Quantification protocol — set up a standard protocol. After completion of the run, open
the Data Analysis window, adjust the data analysis settings in the Quantification tab, and then
click the End Point tab to pick an end point cycle.

n Run an End Point Only protocol — load the End Point Only protocol in the Plate tab of the Run
Setup window, select or create a plate, and start the run

The End Point tab displays the average RFU values to determine whether the target was amplified
by the last (end) cycle. Use these data to determine whether a specific target sequence is present
(positive) in a sample. Positive targets have higher RFU values than the cutoff level you define.

Tip: To create an end point protocol, open the Protocol tab (Run Setup window) and select
Run > End Point Only Run.

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Chapter 9 Data Analysis Details

When the run completes, the data file opens to the End Point tab, which comprises the following
sections:

n Settings — adjusts data analysis settings.

n Results — displays the results immediately after you adjust the settings.

n Well Selector — selects the wells with the end-point data you want to show.

n RFU spreadsheet — displays the end RFU collected in the selected wells.

Results Data
The Results section displays the following data:

n Lowest RFU value — lowest RFU value in the data

n Highest RFU value — highest RFU value in the data

n Negative Control Average — average RFU for the wells that contain negative controls

n Cut Off Value — calculated by adding the tolerance (RFU or Percentage of Range listed in the
Settings) and the average of the negative controls. Samples with RFUs that are greater than
the cutoff value will be called “Positive.” To adjust the cutoff value, change the RFU or
Percentage of Range

The Cut Off Value is calculated using this formula:

Cut Off Value = Negative Control Average + Tolerance

Select a tolerance by one of these methods:

n RFUs (default) — select this method to use an absolute RFU value for the tolerance. The
minimum RFU tolerance value is 2. The maximum is the absolute value of the highest RFU
value minus the absolute value of the lowest RFU value. The default RFU tolerance value is
10% of the total RFU range.

n Percent of Range — select this method to use a percentage of the RFU range for the
tolerance. The minimum percent of range is 1%. The maximum percent of range is 99%. The
default percent of range is 10%.

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 End Point Tab

Adjusting the End Point Data Analysis

To adjust the data in the End Point tab

u Do any of the following:

n Choose a fluorophore from the dropdown list.

n Choose an End Cycle to Average value to set the number of cycles with which to calculate
the average end-point RFU.

n Select RFUs to view the data in relative fluorescence units.

n Select Percentage of Range to view the data as a percentage of the RFU range.

n Select wells in the well selector to focus on subsets of the data.

n Select a well group to view and analyze a subset of the wells in the plate. Select each well
group by name in the Well Group dropdown menu in the toolbar.

RFU Spreadsheet for End Point Analysis

Table 23 defines the data that appear in the RFU spreadsheet in the End Point tab.

Table 23. End Point spreadsheet contents

Information Description
Well Well position in the plate

Fluor Fluorophore detected

Content A combination of the Sample type and Replicate #

End RFU RFU at the end-point cycle

Call Positive or Negative, where positive samples have an RFU value greater
than the average RFU of the negative controls plus the Cut Off Value

Sample Sample Name loaded in the Plate Editor

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Chapter 9 Data Analysis Details

Allelic Discrimination Tab

The Allelic Discrimination tab assigns genotypes to wells with unknown samples. Use these data
to identify samples with different genotypes, including Allele 1, Allele 2, Heterozygote, No Call (no
amplification), or Undetermined.

Note: The data for allelic discrimination must come from multiplex runs with at least two
fluorophores. Each fluorophore identifies one allele in all samples.

Allelic discrimination analysis requires the following minimal well contents:

n Two fluorophores in each well

n NTC (no template control) samples for optimized data analysis

CFX Maestro software offers four options in which to view allelic discrimination data:

n Allelic Discrimination chart — displays the data in a graph of RFU for Allele 1/Allele 2. Each
point in the graph represents data from both fluorophores in one well. You can toggle between
Cartesian and Polar coordinates by selecting and clearing the Polar Coordinates checkbox.
Cartesian Coordinates represents RFU for Allele 1 on the x-axis and RFU for Allele 2 on the
y-axis. Polar Coordinates represents the angle on the x-axis and the RFU distance on the
y-axis from the origin (median of all NTC).

n Well spreadsheet — displays the allelic discrimination data collected in each well of the plate.

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 Allelic Discrimination Tab

n Well selector — selects the wells with the allelic data you want to show.

n Selected Fluorophores panel — changes the x- and y-axis labels in the Allelic Discrimination
chart, the cycle to analyze, and whether to display the call map.

Adjusting Data for Allelic Discrimination

The software automatically assigns a genotype to wells with unknown samples based on the
positions of the NTCs and the angle and distance of the unknown data points from the NTCs.

To adjust allelic discrimination data

u Do any of the following:

n To display polar coordinates, select the checkbox in the Allelic Discrimination chart.

n To view another fluorophore, choose it from the dropdown list in the Selected
Fluorophores panel.

n To change a call, drag across the data point(s) in the Allelic Discrimination chart and
choose an option in the Selected Wells list:

o Allele 1

o Allele 2

o Heterozygote

o Undetermined

o No Call

o Auto Call

Tip: Select Auto Call to revert to the default call.

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Chapter 9 Data Analysis Details

Chart Menu Options

In addition to the common right-click menu options to copy, print, and export charts, Table 24 lists
the menu options available on the Allelic Discrimination chart.

Table 24. Allelic Discrimination chart right- and left-menu options

Menu Option Function

Zoom Focuses in the selected area when more than one data point is
selected.

Well Displays only this well, removes this well from view, sets color for
this trace, or excludes this well from analysis when clicked on a
single trace.

Selected Wells Displays only these wells, removes these wells from view, sets
color for these traces, or excludes these wells from analysis when
multiple traces are selected.

Allelic Discrimination Spreadsheet

Table 25 defines the data that appear in the Allelic Discrimination spreadsheet.

Table 25. Allelic Discrimination spreadsheet contents

Information Description
Well Well position in the plate

Sample Sample name description

Call Identity of the allele, including automatic Allele 1, Allele 2,

Heterozygote, No Call, or Undetermined

Type Auto (Automatic) or Manual, describes how the call was made.
Automatic indicates that the software selected the call. Manual
indicates that the user chose the call

RFU1 RFU for Allele1

RFU2 RFU for Allele2

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 Custom Data View Tab

Custom Data View Tab

The Custom Data View tab simultaneously displays multiple panes in a customizable format.

The Load a Preset View dropdown list offers a selection of display format templates. The default
view displayed is dependent on the file being analyzed. For example, if Melt Curve data are
present, the Amp+Melt default view appears.

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Chapter 9 Data Analysis Details

Creating a Custom Data View

To create a custom data view

u Do any of the following:

n Select an alternate preset view from the dropdown list.

n Select another chart view from the dropdown list located at the top of each individual
pane.

n Change the number of rows and columns in the tab.

n Change individual pane dimensions. Drag the bars at the periphery of each pane.

Click Save as Preset to save the customized as a preset template. Click Manage Presets to delete,
rename, or restore existing preset views.

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 QC Tab

QC Tab
Use the QC tab to quickly assess the quality of the run data based on the rules defined in the QC
tab in the User Preferences window.

CFX Maestro software offers four options in which to view the QC data:

n Amplification chart — displays the RFU for each well at every cycle. Each trace in the chart
represents data from a single fluorophore in one well.

n QC rules table — displays the available QC rules and the settings that define each rule.
Applied QC rules are indicated by a checkmark.

n Well selector — selects the wells with the fluorescence data you want to show.

n QC rule summary pane — displays the selected QC rule and highlights wells that fail the rule.

Changing QC Criteria
To change QC criteria

u Select or clear the Use checkbox for the rule to include or exclude from QC.

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Chapter 9 Data Analysis Details

Excluding Wells That Fail QC

CFX Maestro software displays wells that fail QC criteria in the Results column in the QC rules
table and in the summary pane.

To exclude wells that fail QC criteria

u Select Exclude Wells for each well to exclude.

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 Run Information Tab

Run Information Tab

The Run Information tab displays the protocol and other information about each run. Use this tab to
do the following:

n View the protocol.

n Enter or edit notes about the run.

n Enter or edit the ID or bar code for the run.

n View events that might have occurred during the run. Use these messages to help
troubleshoot a run.

Tip: Right-click the Protocol to copy, export, or print it. Right-click the Notes, ID/Bar Code, or
Other panes to undo, cut, copy, paste, delete, or select the text.

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Chapter 9 Data Analysis Details

Data Analysis Reports

The Report dialog box displays information about the current data file in the Data Analysis window.
To open a report, select Tools > Reports or click Reports on the toolbar.

The Report dialog comprises the following sections:

n Menu and toolbar — provides options to format, save, and print the report or template.

n Options list (top left side of the dialog box) — provides options to display in the report.

n Options pane (bottom left side of the dialog box) — displays text boxes in which you can enter
information about a selected option.

n Preview pane (right side of the dialog box) — displays a preview of the current report.

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 Data Analysis Reports

Data Analysis Report Categories

Table 26 lists all the options available for a data analysis report, depending on the type of data in
the Data Analysis window.

Table 26. Data analysis report categories in the options list

Category Option Description

Header

Title, subtitle, and logo for the report

Report Information Run date, user name, data file name,

data file path, and selected well group

Audit Information Supplementary information required for

auditing, including signatures

Notes Notes about the data report

Run Setup

Run Information Run date, user name, data file name,

data file path, and selected well group

Protocol Text view of the protocol steps and

options

Plate Display Plate view of the information in each

well of the plate

Quantification

Analysis Settings Data collection step number, analysis

mode, and baseline subtraction method

Amplification Chart Amplification chart for runs that include

quantification data

Standard Curve Chart Standard curve chart

Data Spreadsheet listing the data in each

well

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Chapter 9 Data Analysis Details

Table 26. Data analysis report categories in the options list, continued

Category Option Description

Gene Expression — Bar Chart

Analysis Settings Analysis mode, chart data, scaling

option, and chart error

Chart Copy of the bar chart

Target Names Chart of target names

Sample Names Chart of sample names

Data Spreadsheet listing the data in each

well

Target Stability Chart of the target stability values

Box-and-Whisker Chart Box-and-whisker chart

Dot Plot Chart Dot plot chart

Gene Expression — Clustergram, Scatter Plot, Volcano Plot, and Heat Map

Analysis Settings Settings for each chart type

Chart Copy of the chart

Data Spreadsheet listing the data in each

target

Gene Expression — ANOVA Data

ANOVA Settings P-value threshold used in the analysis

ANOVA Results Table of results from ANOVA and

Tukey's HSD post-hoc analysis

Shapiro-Wilk Normality Test Biological group, count, P-value, and

errors that occur for each target in the
analysis

ANOVA Errors Errors identified during ANOVA

calculations

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Table 26. Data analysis report categories in the options list, continued

Category Option Description

Melt Curve

Analysis Settings Melt step number and threshold bar

setting

Melt Curve Chart Melt curve chart

Melt Peak Chart Melt peak chart

Data Spreadsheet listing the data in each

well

Allelic Discrimination

Analysis Settings Displays fluorophores, cycle, and view

call map

Allelic Discrimination Chart Copy of the allelic discrimination chart

Data Spreadsheet listing the data in each

well

End Point

Analysis Settings Fluorophore, end cycles to average,

mode, lowest RFU value, highest RFU
value, and cut off value

Data Spreadsheet listing the data in each

well

QC Parameters

Data Spreadsheet listing the parameters for

each QC rule

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Chapter 9 Data Analysis Details

Creating a Data Analysis Report

You can save the report layout as a template, which you can use again for similar reports.

To create a data analysis report

1. Make final adjustments to the well contents, selected wells, charts, and spreadsheets in the
Data Analysis window before creating the report.

2. Select Tools > Reports in the Data Analysis menu bar to open the Report dialog box.

3. Choose the options you want to include in the report. The report opens with default options
selected. Select or clear the checkboxes to change whole categories or individual options
within a category.

Note: The data that appear in the report depend on the current selections within the tabs
of the Data Analysis or window. For example, a quantification run might not contain a
standard curve, and therefore those data do not appear in the Data Analysis window or in
the data report.

4. Change the order of categories and items in a report. Drag the options to the relative position.
Items can be reordered only within the categories to which they belong.

5. (Optional) In the Report Options pane, enter information relevant to the selected option:

n Choose a subset of information to display in the report.

n Choose specific settings for the selected option.

n Change the text to display for the selected option.

6. Click Update Report to update the Report Preview with any changes.

7. Print or save the report. Click the Print Report button in the toolbar to print the current report.
Select File > Save to save the report in PDF (Adobe Acrobat Reader file), MHT (Microsoft
document), or MHTML (Microsoft document) file format and select a location in which to save
the file. Select File > Save As to save the report with a new name or in a new location.

8. (Optional) Create a report template with the information you want. To save the current report
settings in a template, select Template > Save or Save As. Then load the report template the
next time you want to make a new report.

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Creating Well Group Reports

To create a well group report

1. Select Tools > Well Group Reports in the Data Analysis window.

2. In the Well Groups Reports dialog box, select the well groups, amplification steps, and melt
steps to include in the report.

3. Enter the path or navigate to the destination folder in which to save the report.

4. (Optional) Select Choose a Report Template and navigate to the template file folder.

5. (Optional) Select Open Destination Folder to open the folder and view the reports after they
are generated.

6. Click Create Reports.

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Chapter 9 Data Analysis Details

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Chapter 10 Gene Expression Analysis
With the use of stringently qualified controls in your reactions, you can perform a gene expression
run to normalize the relative differences in a target concentration among samples. Typically,
expression levels for one or more reference genes are used to normalize the expression levels of
a gene of interest. Reference genes take into account loading differences or other variations
represented in each sample and their expression levels should not be affected in the biological
system being studied.

Choose the Gene Expression tab in the Data Analysis window to evaluate relative differences
between PCR reactions in two or more wells. For example, you can evaluate relative numbers of
viral genomes or relative numbers of transfected sequences in a PCR reaction. The most common
application for gene expression study is the comparison of cDNA concentration in more than one
reaction to estimate the levels of steady state messenger RNA.

The software calculates the relative expression level of a target with one of these scenarios:

n Relative expression level of a target sequence (Target 1) relative to another target (Target 2);
for example the amount of one gene relative to another gene under the same sample
treatment.

n Relative expression level of one target sequence in one sample compared to the same target
under different sample treatment;. for example, the relative amount of one gene relative to
itself under different temporal, geographical, or developmental conditions.

Plate Setup for Gene Expression Analysis

To perform gene expression analysis, the contents of the wells must include the following:

n Two or more targets — the two targets that represent different amplified genes or sequences in
your samples.

n One or more reference targets — at least one target must be a reference target for normalized
expression. Assign all reference targets in the Experiment Settings window to analyze the data
in Normalized Expression mode (ΔΔCq ). Runs that do not contain a reference must be
analyzed using Relative Expression mode (ΔCq ).

n Common samples — your reactions must include common samples (minimum of two required)
to view your data plotted in the Gene Expression tab. These samples should represent

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Chapter 10 Gene Expression Analysis

different treatments or conditions for each of your target sequences. Assign a control sample
(optional) in the Experiment Settings window. If no control is selected, the software uses the
lowest Cq as the control.

The requirements for Gene Expression setup in the Plate Editor depend on whether reaction
contents are singleplex PCR, with one fluorophore in the reactions, or multiplex PCR, with more
than one fluorophore in the reactions.

Guided Plate Setup

If the plate setup of a data file does not contain the information required for analysis and the Gene
Expression tab is selected, the space normally occupied by the bar chart will contain instructions
for entering this information. For normalized gene expression, complete the following steps:

1. Define Target and Sample names using any of the following:

n Plate Setup — opens the Plate Editor window.

n Replace Plate File — opens the Select Plate browser, in which you can navigate to a
previously saved plate file with which to replace the current plate layout.

n Replace PrimePCR File — opens the Select PrimePCR file dialog box, in which you can
navigate to a PrimePCR™ run file and apply it to the plate layout.

2. Select one or more reference targets and a control sample using the Experiment Settings
dialog box.

If the plate layout already contains target and sample information, only the second step is
required and is highlighted orange. This step must be completed before normalized gene
expression analysis can occur.

Note: Data for the clustergram, scatter plot, heat maps, and volcano plot are displayed
only if all of the requirements for normalized gene expression listed under Plate Setup for
Gene Expression Analysis are met.

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Gene Expression Charts

CFX Maestro™ software displays gene expression data in multiple views. Table 27 lists chart
options available in the software.

Table 27. Gene expression chart options

Button Name Function

Graphing Displays normalized gene expression data
in one of the following views:
n Bar chart (the default)
n Box and whisker chart
n Dot plot chart

Clustergram Displays the normalized expression data in

a hierarchy based on the degree of similarity
of expression for different targets and
samples.

Scatter Plot Displays the normalized expression of

targets for a control versus an experimental
sample.

Volcano Plot Displays the relative expression of targets

in the selected biological group as
compared to the control group and indicates
the degree of significance based on the P-
value.

Heat Map Displays a visual depiction of the regulation

of targets for an experimental sample
compared to a control sample based on
relative normalized expression and its
location on a plate.

ANOVA Displays the results of one-way ANOVA on

the gene expression data using the following
R packages to perform ANOVA and
determine Tukey results:
n Companion to Applied Regression (car)
n Least-square means (lsmeans)

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Chapter 10 Gene Expression Analysis

Table 27. Gene expression chart options, continued

Button Name Function

Reference Gene Selection Tool (Available on the Study Analysis tab in the
Gene Study window) Identifies the tested
reference genes and categorizes them as
Ideal, Acceptable, or Unstable based on
their stability.

PrimePCR Controls Analysis (Available on the Study Analysis tab in the

Gene Study window) Displays the results of
the tested samples.

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Graphing

The relative expression of targets is presented in these two views:

n Gene Expression chart — displays the real-time PCR data as one of the following:

o ΔΔCq — relative normalized expression calculated using control samples and reference
targets.

o ΔCq — relative quantity of the target gene in a sample relative to a control sample.

You can view the data in the gene expression chart in one of three views. See Changing and
Annotating the Chart View on page 195 for more information.

n Spreadsheet — displays a spreadsheet of the gene expression data.

Tip: Right-click any chart or spreadsheet for options. Select View/Edit Plate from the Plate
Setup dropdown menu to open the Plate Editor and change well contents in the plate.

Tip: Select Sort from the right-click menu to rearrange the order of the Target and Sample
names in the chart.

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Chapter 10 Gene Expression Analysis

Normalized Gene Expression

To normalize data, use the measured expression level of one or more reference genes as a
normalization factor. Reference genes are targets that are not regulated in the biological system
being studied, such as actin, GAPDH, or tubulin.

To set up normalized gene expression (ΔΔCq) analysis

1. Open a data file (.pcrd extension).

2. Review the data in the Quantification tab of the Data Analysis window. Make adjustments to
the data, such as changing the threshold and the analysis mode.

3. Choose the Gene Expression tab.

4. In the Gene Expression tab, click Experiment Settings.

5. In the Experiment Settings dialog box, do the following:

a. Choose the Samples tab and select a control. When a control is assigned, CFX Maestro
software normalizes the relative quantities for all genes to the control quantity, which is set
to 1.

b. Choose the Target tab and select reference genes. Gene expression analysis requires
one reference among the targets in your samples.

6. Select Normalized Expression (ΔΔCq ) if it is not already selected, and then view the
expression levels in the Gene Expression tab.

Note: You can also use the Setup Wizard to set up the plate layout for normalized gene
expression analysis.

Relative Quantity

By definition, relative quantity (ΔCq ) data are not normalized. This method is used to quantitate
samples that do not include any reference genes (targets). Typically, researchers are confident in
one of the following considerations when they set up their run:

n Each sample contains the same amount of template, possibly the same mass of RNA or cDNA
in each well.

n Any variance in the amount of biological sample loaded will be normalized after the run by
some method in the data analysis outside of the software. For example, a researcher might
choose to simply divide the relative quantity value by the normalizing factor, possibly the mass
of nucleic acid loaded for each sample, or the number of cells from which the nucleic acid was
isolated.

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To run a Relative Quantity (ΔCq) analysis

u In the Gene Expression tab, select Relative Quantity (ΔCq ) from the Mode dropdown list in the
right pane.

Tip: To compare results to data from other gene expression runs, open a new gene study
or add a data file to an existing gene study.

Changing and Annotating the Chart View

Using the charts toolbar menu commands and the data analysis chart tools, you can change the
chart view, annotate each chart, and change the chart display. The charts toolbar appears between
the chart and the data analysis spreadsheet at the bottom of the screen.

Chart Toolbar Tools

Tip: See Charts on page 138 for information about the chart tools that appear on the right side
of data analysis charts.

The toolbar below the charts provides quick access to annotation tools.

Table 28 lists the functions of the buttons in the charts toolbar.

Table 28. Charts toolbar

Button Name Function

Bar chart Displays the relative expression of the targets.

Box and Whisker chart Displays data as quartile ranges (see Box and
Whisker Chart Calculations on page 241 for
calculation details).
Note: Available only if Analyze Using is set to
Biological Groups Only.

Dot Plot chart Displays the individual sample data points for each
target.
Note: Available only if Analyze Using is set to
Biological Groups Only.

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Chapter 10 Gene Expression Analysis

Table 28. Charts toolbar, continued

Button Name Function

Add Arrow Draws an arrow on the active chart.

Add Circle Draws a circle on the active chart

Add Text Inserts a textbox on the active chart, in which you can
add text to identify items of interest in the chart.

Undo Removes or reverts the last annotation performed on

the active chart.

Redo Reverts the last Undo action performed on the active

chart.

Clear All Clears all annotations on the active chart.

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Sorting Target, Sample, and Biological Group Data

Note: This option is available on gene expression charts only.

By default, the Targets, Samples, and Biological Groups lists appear in alphabetical order. Use the
Sort dialog box to sort the display in reverse alpha order or to manually move a term to a different
position in the list.

To sort target, sample, and biological group data

1. From the chart tools, click Sort.

The Gene Expression Chart Sorting dialog box appears.

2. In the dialog box, click Z-A to sort the list in reverse alphabetical order.

3. To manually move a term, select it and click the appropriate button between the charts:

n Click the Up or Down arrow to move the selected term one position.

n Click the Up or Down bar arrow to move the selected term to the top or bottom of the list.

4. Click OK to save the changes and return to the Gene Expression tab.

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Chapter 10 Gene Expression Analysis

Changing the Target, Sample, and Biological Group Color Settings

Use the Color Settings dialog box to change the color of a target, sample, or biological group, or
remove the item from the graph.

To change the target color settings

1. In the right pane in the Gene Expression dialog box, verify that Sample appears in the X-Axis
dropdown list.

2. In the Chart Tools, select Color Settings.

The Color Settings dialog box appears.

3. To change the display color for a target, click its color in the Color column.

4. In the Color dialog box that appears, select a new color and click OK.

5. To remove a target from the gene expression graph, clear its checkbox in the Show Chart
column.

Tip: To clear all targets, clear Show Chart in the column head.

6. (Optional) By default, the bars appear in solid colors. To display the bars in gradient colors,
clear Use Solid Colors.

7. Click OK to save the changes and return to the Gene Expression tab.

To change the sample or biological group color settings

1. In the right pane on the Gene Expression dialog box, verify that Target appears in the X-Axis
dropdown list.

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2. Perform the steps in To change the target color settings on page 198.

Changing the Chart View

To change the current chart view

u Select the toolbar menu command for the target view.

Note: The Gene Expression tab always opens displaying the data in default Bar Chart view.

Excluding Outlier Data Points

In the Dot Plot chart, you can easily view and exclude outliers from your analysis.

To exclude outlier data points

u In the Dot Plot chart, right-click the target outlier and select Exclude Well from Analysis.

The data point is removed from the Dot Plot chart and the well changes to gray in the Well Selector
in the Quantification tab.

To include an excluded outlier data point

u In the Quantification tab, right-click the well in the Well Selector and select Well > Include in
Analysis.

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Chapter 10 Gene Expression Analysis

Viewing Data Point Details

To view data point details

u In the Box and Whisker plot or the Dot Plot, pause your cursor on an individual data point.

A tooltip displays, showing the sample name and its expression (relative quantity or normalized
expression, depending on the selected mode).

Annotating Charts

You can add arrows, circles, and text to each bar chart view to clearly communicate data. The
annotations are saved with the bar chart and appear in the exported and printed file. However,
annotations made to one chart view are not added to the other chart views.

To draw an arrow or circle on the chart

1. In the bar chart toolbar, click the specific tool.

2. Click in the bar chart and drag your cursor across the chart as necessary.

To add text to the chart

1. In the bar chart toolbar, click Add Text.

2. Click in the bar chart. A text box appears at that location.

3. Add text in the text box.

4. Click anywhere on the chart to exit the text box.

Tip: Press Enter to add multiple lines to the text box.

To move an annotation

1. Hover your cursor over the annotation. The icon changes to a pointing finger and the
annotation border is highlighted.

2. Click the annotation and drag it to another position.

3. Release the annotation to secure its position.

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To undo an annotation

u Click Undo.

The most recently added annotation is removed.

Tip: You can undo the ten most recent annotations, one at a time.

To redo an annotation

u Click Redo.

The most recently removed annotation returns.

Tip: You can redo the ten most recent annotations, one at a time.

To delete an annotation

u Right-click the annotation and select Delete.

Adjusting Gene Expression Data

After selecting your analysis mode, adjust the data you view in the Gene Expression tab by
changing the settings options to the right of the chart.

Graph Data

Set the y-axis value to Linear scale to enable graph data options. Graph data options allow you to
present the data in the graph with one of these options:

n Relative to control — graph the data with the axis scaled from 0 to 1. If you assign a control in
your run, select this option to quickly visualize upregulation and downregulation of the target.

n Relative to zero — graph the data with the origin at zero.

Tip: You set the graph data options in the Gene Expression tab in the User Preferences dialog box.

Analyze Using

Use the dropdown menu to select how data is analyzed and plotted. The options are:

n Samples Only — data are analyzed and plotted on a per sample basis.

n Biological Groups Only — data are analyzed and plotted for biological groups. The expression
displayed for the biological group is the geometric mean of the samples in that group.

n Sample Biological Group — data are analyzed and plotted on a per sample basis with the
biological group appended after the sample name. The P-values shown are calculated based
on the biological group.

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Chapter 10 Gene Expression Analysis

n Biological Group Sample — data are analyzed and plotted on a per sample basis with the
biological group prepended before the sample name. The P-values shown are calculated
based on the biological group.

Control

Use the Control dropdown menu to select a sample or biological group (depending on your
Analyze Using selection) that will be used to normalize the Relative Quantity:

n If you selected Samples Only, the Control dropdown list displays all samples in the study.

n If you selected Biological Groups Only, Sample Biological Group, or Biological Group Sample,
the Control dropdown list displays all biological groups in the study.

Note: The selected biological group is also used to calculate the P-value.

Tip: When analyzing by samples only or biological groups only, setting Control to None
enables the Scaling option, in which you can scale the data to Average, Lowest, Highest, or
Unscaled. See Scaling Options on page 203 for more information.

Annotate P-Values and P-Value Threshold

When Annotate P-Values is selected, the software displays an asterisk (*) on the bar chart above a
target if its P-value is below the selected threshold. The software automatically calculates the P-
value by comparing the sample’s expression level to the selected control sample’s expression
level using a standard t-test. The P-value threshold range is 0.000—1.000.

X-Axis Options

The x-axis option allows you to select the x-axis data of the Gene Expression chart:

n Target — graphs the target names on the x-axis.

n Sample — graphs the sample names on the x-axis.

Y-Axis Options

The y-axis option allows you to show the Gene Expression chart in one of these three scales:

n Linear — select this option to show a linear scale.

Tip: Setting the y-axis to Linear enables the Graph Data dropdown list, from which you
can choose to graph data relative to control or relative to zero.

n Log 2 — select this option to evaluate samples across a large dynamic range.

n Log 10 — select this option to evaluate samples across a very large dynamic range.

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Scaling Options

Select Normalized Gene Expression (ΔΔCq ) and set the Control to None (for either Sample or
Biological Group) to enable the scaling options in the Gene Expression chart. Select one of these
scaling options to calculate and present your data in a manner that best suits your run design:

n Unscaled — presents the unscaled normalized gene expression.

n Highest — scales the normalized gene expression for each target by dividing the expression
level of each sample by the highest level of expression in all the samples.

This scaling option uses the scaled-to-highest formula.

n Lowest — scales the normalized gene expression for each target by dividing the expression
level of each sample by the lowest level of expression in all the samples.

This scaling option uses the scaled-to-lowest formula.

n Average — scales the normalized gene expression for each target by dividing the expression
level of each sample by the geometric mean of the expression levels for all the samples.

This scaling option uses the scaled-to-average formula.

Error Bar

Select an option for the type of error calculations (error bars) in the Gene Expression chart:

n Options if Analyze Using is set to Samples Only, Sample Biological Group, or Biological Group
Samples:

o Standard error of the mean (default)

o Standard deviation

n Options if Analyze Using is set to Biological Groups Only:

o Confidence interval (default)

o Standard error of the mean (Log2 expression)

o Standard deviation (Log2 expression)

Chart Error Bar Multiplier

Select a multiplier for the error bars in the Gene Expression chart. Select one of these integers:
+/- 1 (default), 2, or 3. The type of multiplier changes when you select the error bar:

n SEMs for standard error of the mean

n Std Devs for standard deviations

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Chapter 10 Gene Expression Analysis

Experiment Settings
Tip: This dialog box is also available in the Plate Editor. For more information, see Changing
Experiment Settings on page 95.

In the Experiment Settings dialog box, you can view or change the list of targets and samples or
biological groups, select reference genes, select controls, or set the Gene Expression Analysis
group to be analyzed if biological groups have been added to the wells.

To open the Experiment Settings dialog box

u In the Graphing tab, click Experiment Settings at the bottom of the right pane.

The Experiment Settings dialog box appears displaying the Targets tab.

To adjust Targets settings

u In the Targets tab, do any of the following:

n To select a target as a reference for gene expression data analysis, select its name in the
Reference column.

n To change the target’s color, click its cell in the Color column and change the color in the
Color dialog box that appears.

The color change appears in the Gene Expression charts.

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n To use a previously determined efficiency value, clear the target’s checkbox in the Auto
Efficiency column and enter a number for the efficiency percentage of a target.

The software calculates the relative efficiency for a target using Auto Efficiency if the data
for a target include a standard curve.

To adjust the Sample or Biological Group settings

u In the Samples and Biological Groups tab, do any of the following:

n To select a sample as a control for gene expression data analysis, select its name in the
Control column.

n To select a biological group as a control for gene expression data analysis, select its
name in the Control column.

n To change the sample or biological group’s color, click its cell in the Color column and
change the color in the Color dialog box that appears.

The color change appears in the Gene Expression charts.

n To display the sample or biological group in the Gene Expression charts, select it in the
Show Chart column.

n To remove the sample or biological group from the Gene Expression charts, clear it in the
Show Chart column.

Tip: The sample or biological group’s data remain in the Results table.

To exclude a sample type from analysis calculations

u Select its checkbox at the bottom of the Experiment Settings dialog box.

Note: This excludes controls and/or standards from gene expression analysis.

Right-Click Menu Options

Right-click on the gene expression chart to select the items shown in Table 29.

Table 29. Gene expression right-click menu items

Item Function
Copy Copies the chart to a clipboard.

Save Image As Saves the chart as an image file. Set the resolution and dimensions
of the image and then select the file type (PNG, JPG, or BMP).

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Table 29. Gene expression right-click menu items, continued

Item Function
Page Setup Selects a page setup for printing.

Print Prints the chart.

Set Scale to Default Show All displays all of the data in the bar chart. Scroll Bar
displays a scroll bar if there are too many samples to display in the
chart frame while maintaining a minimum bar width.

Chart Settings Opens the Chart Settings window to adjust the graph.

Sort Sorts the order of samples or targets that appear on the chart x-axis.

Color Settings Opens the Color Settings window, from which you can change the
color of targets or samples in the chart.

User Corrected Std Devs Calculates the error bars using the corrected standard deviation
formula.

Use Solid Bar Colors Displays solid bars in the chart.

X–Axis Labels Displays x-axis labels horizontally or angled.

Data Spreadsheet
Table 30 defines the data displayed in the Gene Expression Data Table.

Note: The values in the table are calculated based on the graph type and preferences
selected in the right-hand pane.

Table 30. Description of information in the spreadsheet on the Graphing tab

Information Description
Target Target name (amplified gene) selected in the Experiment Settings
window.

Sample Sample and/or biological group name selected in the Experiment

Biological Group Settings window.

Sample Biological Group

Biological Group Sample

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Information Description
Control Control name selected in the Experiment Settings window. When
Analyze Using is set to Samples Only, Control is the sample
selected in the Experiment Settings window. When either Biological
Groups Only, Sample Biological Group, or Biological Group
Sample is selected, control is the biological group selected in the
Experiment Settings window.

Relative Quantity or Relative Quantity (ΔCq ) or Normalized Gene Expression (ΔΔCq ),

Expression depending on the selected mode.

Relative Quantity or Standard error of the mean (SEM) or standard deviation (SD) of the
Expression SEM (or SD) relative quantity or normalized expression, depending on the
selected option. Available only if Analyze Using is set to Samples
Only, Sample Biological Group, or Biological Group Sample.

Corrected Relative Quantity or Corrected value calculation for SEM or SD of the relative quantity
Expression SEM (or SD) or normalized expression, depending on the selected option.
Available only if Analyze Using is set to Samples Only, Sample
Biological Group, or Biological Group Sample.

Mean Cq Mean of the quantification cycle (not displayed if Analyze Using is

set to Biological Groups Only).

Cq SEM (or SD) SEM or SD of the quantification cycle, depending on the selected
option (not displayed if Analyze Using is set to Biological Groups
Only).

P-Value A measure of statistical significance derived from the student's

t-test. Refer to P-Value on page 229 for additional information.
Note: The P-values reported in the bar chart are based on the
selected control. You can choose to annotate the chart with P-
values falling below a user-specified threshold.

Relative Quantity n% CI Low Confidence interval (CI) lower limit, where the % value is based on
or the P-value threshold. Selected Mode determines whether it is
Expression n% CI Low relative quantity or expression. Available only if Analyze Using is
set to Biological Groups Only and Error Bar is set to Confidence
Interval.

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Information Description
Relative Quantity n% CI High Confidence interval (CI) upper limit, where the % value is based on
or the P-value threshold. Selected Mode determines whether it is
Expression n% CI High relative quantity or expression. Available only if Analyze Using is
set to Biological Groups Only and Error Bar is set to Confidence
Interval.

RQ Lower Error Bar Value of lower error bar when Error Bar is set to Standard
or Deviation(lg) or Standard Error of the Mean(lg). Selected
Exp. Lower Error Bar Mode determines whether it is relative quantity (RQ) or
expression (Exp.). Available only if Analyze Using is set to
Biological Groups Only.

RQ Upper Error Bar Value of upper error bar when Error Bar is set to Standard
or Deviation(lg) or Standard Error of the Mean(lg). Selected Mode
Exp. Upper Error Bar determines whether it is relative quantity (RQ) or expression (Exp.).
Available only if Analyze Using is set to Biological Groups Only.

Show Details Option

Table 31 defines the data displayed when Show Details is selected from the right-click menu of the
bar chart spreadsheet.

Table 31. Information in the bar chart spreadsheet with Show Details selected

Information Description
Data Set Fluorescence data from one fluorophore in the data file

Relative Quantity Calculated relative quantity of samples

Relative Quantity SD Standard deviation of the relative quantity calculation

Corrected Relative Quantity SD Calculated standard deviation of the corrected relative quantity

Relative Quantity SEM Standard error of the mean of the relative quantity calculation

Corrected Relative Quantity Calculated standard error of the mean of the corrected relative
SEM quantity

Relative Quantity(lg) Log2 of the relative quantity that is used for statistical analysis

SD RQ(lg) Standard deviation of the relative quantity (log2 )

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Table 31. Information in the bar chart spreadsheet with Show Details selected, continued

Information Description
SEM Expression(lg) Standard error of the mean of the expression (log2 )

Unscaled Expression Calculated unscaled expression

Unscaled Expression SD Calculated standard deviation of the unscaled expression

Corrected Unscaled Calculated standard deviation of the corrected unscaled expression

Expression SD

Unscaled Expression SEM Calculated standard error of the mean of the unscaled expression

Corrected Unscaled Calculated standard error of the mean of the corrected unscaled
Expression SEM expression

Unscaled Expression(lg) Log2 of the unscaled expression

SD Unscaled Expression(lg) Standard deviation of the unscaled expression (log2 )

SEM Unscaled Expression(lg) Standard error of the mean of the unscaled expression (log2 )

Expression Normalized gene expression

Corrected Expression SD Calculated standard deviation

Expression SEM Standard error of the mean

Corrected Expression SEM Calculated standard error of the mean

Expression(lg) Log2 of the expression (normalized expression) that is used for

statistical analysis

SD Expression(lg) Standard deviation of the expression (log2 )

SEM Expression(lg) Standard error of the mean of the expression (log2 )

Sample # Number of samples in the biological group

Mean Cq Mean of the quantification cycle (not displayed when Analyze Using
is set to Biological Groups Only)

Cq SD Standard deviation of the quantification cycle (not displayed when

Analyze Using is set to Biological Groups Only)

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Chapter 10 Gene Expression Analysis

Table 31. Information in the bar chart spreadsheet with Show Details selected, continued

Information Description
Cq SEM Standard error of the mean of the quantification cycle (not displayed
when Analyze Using is set to Biological Groups Only)

P-Value A measure of statistical significance derived from the student's

t-test. Refer to P-Value on page 229 for additional information.
Note: The P-values reported in the bar chart are based on the
selected control. You can choose to annotate the chart with P-
values falling below a user-specified threshold.

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 Clustergram

Clustergram
The clustergram displays the data in a hierarchy based on the degree of similarity of expression for
different targets and samples.

Note: You must choose a reference target to display any of the data plots other than relative
expression for bar charts.

The clustergram image depicts relative expression of a sample or target as follows:

n Upregulation (red) — higher expression

n Downregulation (green or blue) — lower expression

n No regulation (black)

n No value calculated (black with a white X)

The lighter the shade of color, the greater the relative expression difference. If no normalized Cq
value can be calculated, the square will be black with a white X.

On the outer edges of the data plot is a dendrogram, which indicates the clustering hierarchy.
Targets or samples that have similar expression patterns will have adjacent branches while those
with dissimilar patterns will be more distant.

Settings
You can set the following options:

n Cluster By — choose from Targets, Samples, Both, or None.

n Size — adjusts the image size and changes the degree of chart magnification for easier
visualization.

n Split Out Replicates — displays values for the individual replicates.

Tip: You can change the color scheme for clustergram, scatter plot, heat map, and volcano
plot from the default Red/Green to Red/Blue by selecting this option from the right-click menu
on any of these charts.

Right-Click Menu Options

Right-click menu options for the clustergram are the same as those for the bar chart. See Table 29
on page 205 for available options. In addition, select Color Scheme to change the downregulation
expression from the default Red/Green to Red/Blue on the chart.

Data Spreadsheet
The spreadsheet displays values for the target, sample, and normalized expression.

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Chapter 10 Gene Expression Analysis

Scatter Plot
The scatter plot displays the normalized expression of targets for a control versus an experimental
sample.

The lines in the plot indicate the fold change threshold. Data points between the lines indicate that
the difference in expression for that target (gene) is negligible between the samples. Data points
outside the lines exceed the fold change threshold and might be of interest.

The plot image shows the following changes in target expression based on the fold change
threshold:

n Upregulation (red circle) — relatively higher expression

n Downregulation (green or blue circle) — relatively lower expression

n No change (black circle)

Click and drag either threshold line to adjust the fold change threshold value.

Settings
You can set the following options:

n Control Sample

n Experimental Sample

n Fold Change Threshold

As you increase or decrease the fold change value, the threshold lines in the plot move
accordingly.

Right-Click Menu Options

Right-click menu options for the scatter plot are the same as those for the bar chart. See Table 29
on page 205 for available options. In addition, select Symbol to change the symbol used on the
plot from the default circle to one of the following:

n Triangle

n Cross

n Square

n Diamond

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 Scatter Plot

Data Spreadsheet
The spreadsheet displays the values for the target and normalized expression for control and
experimental samples. It also indicates whether targets are up- or downregulated compared to the
target regulation.

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Chapter 10 Gene Expression Analysis

Volcano Plot
The volcano plot displays the relative expression of targets in an experimental biological group
compared to a control and indicates the degree of significance based on P-value.

The lines in the plot indicate the fold change threshold. Data points between the lines indicate that
the difference in expression for that target (gene) is negligible. Data points outside the lines
exceed the fold change threshold and might be of interest.

Note: In the Charts tab, if you choose to analyze using only samples, the Volcano Plot tab is
grayed out (not usable). Ensure that you have selected a control biological group in the
Experiment Settings dialog box and that your analysis includes biological groups in the Charts
tab.

The plot displays the following fold changes based on the threshold setting:

n Upregulation (red circle) — higher expression

n Downregulation (green or blue circle) — lower expression

n No change (black circle)

Click and drag either threshold line to adjust the fold change threshold value.

Settings
You can set the following options:

n Control Biological Group

n Experimental Biological Group

n Fold Change Threshold

n P-Value Threshold

As you increase or decrease Fold Change Threshold or P-Value Threshold values, the
threshold lines in the plot move accordingly.

Right-Click Menu Options

Right-click menu options for the volcano plot are the same as those for the bar chart. See Table 29
on page 205 for available options. In addition, select Symbol to change the symbol used on the
plot from the default circle to one of the following:

n Triangle

n Cross

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 Volcano Plot

n Square

n Diamond

Data Spreadsheet
The spreadsheet displays the values for the target, biological group, fold change, and P-value. It
also indicates whether the P-value exceeds its threshold (significant P-value) and the target is up-
or downregulated compared to the target regulation.

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Chapter 10 Gene Expression Analysis

Heat Map
The heat map presents a visual depiction of the regulation of targets for an experimental sample
compared to a control sample based on relative normalized expression and its location on a plate.

Note: This feature should be used only with singleplex PCR experiments.

A legend below the heat map displays the range of normalized expression, which corresponds to

n Upregulation (red) — higher expression

n Downregulation (green or blue) — lower expression

n No change (black)

The lighter the shade of color, the greater the relative normalized expression difference. If no
normalized expression value can be calculated, the square appears black with a white X.

Settings
You can set the following options:

n Control Sample

n Experimental Sample

n Size — use the slider to adjust the image size to change the degree of chart magnification

n Split Out Replicates — displays values for the individual replicates

Right-Click Menu Options

Right-click menu options for the heat map are the same as those for the bar chart. See Table 29 on
page 205 for available options. In addition, select Color Scheme to change the downregulation
expression from the default Red/Green to Red/Blue on the chart.

Data Spreadsheet
The spreadsheet displays the values for the target, sample, and fold change.

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 ANOVA

ANOVA
The ANOVA tab displays the results of one-way analysis of variance (ANOVA). ANOVA tests the
hypothesis that the mean target expression between any two biological groups is equal. The null
hypothesis states that the mean expression of all biological groups is equal, while the alternative
hypothesis states that at least one biological groups is different.

CFX Maestro software also performs a pairwise comparison of the mean expression between
biological groups via Tukey's HSD test.

CFX Maestro software uses the following R packages to perform ANOVA and to determine the
Tukey's HSD post hoc results:

n car (companion to applied regression)

n lsmeans (least-square means)

Settings
You can set the P-value threshold for one-way ANOVA.

Calculating ANOVA

When you first select the ANOVA tab, click "Not current. Click to recalculate" to calculate one-way
ANOVA. If successful, the data appear in the ANOVA tab in the Results table in the lower pane
(see Results Table on page 218).

Note: If ANOVA cannot be performed, the software automatically displays the Errors tab, which
provides possible reasons for errors reported in the ANOVA analysis.

To recalculate ANOVA

u In the ANOVA tab, click "Not current. Click to recalculate."

The next time you open the file and select the ANOVA tab, CFX Maestro recalculates ANOVA using
the saved settings.

Exporting R Scripts

After calculating ANOVA, you can export the results and the R script, and open the .r file in a
stand-alone R application. The .r script requires the car and lsmeans packages. The exported .r
script contains the code for the required packages and will install them if they are not installed in
the stand-alone R application.

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Chapter 10 Gene Expression Analysis

To export R scripts

u In the ANOVA tab, click Export R Script and provide a name and location for the .r file in the
Save As dialog box that appears.

Results Table
Data from ANOVA appear in tabs in the results table. Table 32 defines the columns for each tab in
the table.

Table 32. ANOVA Results table column descriptions

Tab Heading Description

ANOVA

Target The gene/region of interest that will be amplified by PCR

df The number of values in the final calculation that are

allowed to vary

P-Value The ANOVA P-value calculated for the target. This

ANOVA P-value is not corrected for multiple testing.

Contrast The two biological groups that are being compared

Ratio The ratio of the expression for the two biological groups
compared. The numerator is the first biological group in
Contrast and the denominator is the second biological
group in Contrast.

P-Value BH The ANOVA P-value after Benjamini-Hochberg

correction for false discovery rate.
Note: This column is displayed only if the
experiment contains multiple genes.

Lower The lowest limit value associated with the confidence

Bound (X%) interval that, at a given percent probability, the
confidence interval will contain the true population mean

Upper The highest limit value associated with the confidence

Bound (Y%) interval that, at a given percent probability, the
confidence interval will contain the true population mean

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 ANOVA

Table 32. ANOVA Results table column descriptions, continued

Tab Heading Description

P-Value The P-value derived from Tukey's HSD test, indicating
Tukey the significance of the difference in expression between
the two biological groups tested. This P-value is
corrected for the family-wise error rate.

Significant The significance of the P-value for these conditions:

n For multiple-gene tests, if P-Value BH or P-Value
Tukey shows significance (P-Value BH or P-Value
Tukey is less than 0.05) the Significant column for the
associate ANOVA row displays Yes; otherwise, it
displays No.
n For single-gene tests, if P-Value ANOVA shows
significance (P-Value ANOVA < 0.05) the Significant
column for the associate ANOVA row displays Yes;
otherwise, it displays No.

Shapiro-Wilk The Shapiro-Wilk test is used to determine the

Normality Test likelihood that the expression values of the samples in a
biological group are obtained from a normally distributed
population. The null hypothesis for this test states that
the population of expression values for the biological
group is normally distributed. A smaller P-value
indicates that expression values are less likely to come
from a normal distribution.

Target The gene/region of interest that will be amplified by PCR

Biological The set of samples for which the normality of the

Groups expression values is tested

Count The number of samples in the biological group (must be

between 3 and 5,000)
Tip: Use this column to view balanced vs.
unbalanced datasets.

P-Value The probability that the population of expression values

are normally distributed. Calculated from the W-value

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Chapter 10 Gene Expression Analysis

Table 32. ANOVA Results table column descriptions, continued

Tab Heading Description

Normal Indicates whether the expression of the target is
normally distributed among the samples in the specified
biological group based on the P-value exceeding the
user-defined threshold:
"Yes" if P-Value > P-Value Threshold
"No" if P-Value < P-Value Threshold

Error Errors encountered while performing the Shapiro-Wilk

test

Errors

Message The reason for errors reported in the ANOVA analysis

Right-Click Menu Options

Right-click menu options for the ANOVA results chart are the same as those for the bar chart. See
Table 29 on page 205 for available options.

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 Gene Study

Gene Study
Create a gene study to compare gene expression data from one or more real-time PCR
experiments using an inter-run calibrator to normalize between the experiments. Create a gene
study by adding data from one or more data files (.pcrd extension) to the gene study. The software
groups them into a single file (.mgxd extension).

Note: The maximum number of samples you can analyze in a gene study is limited by the size
of the computer’s RAM and virtual memory.

Inter-Run Calibration
Inter-run calibration is automatically attempted in every gene study for each target to normalize
inter-run variations between targets assayed in separate real-time PCR runs (that is, different .pcrd
files generated from different plates).

For the software to recognize a sample as an inter-run calibrator, it must share the same target
name, sample name, and, if used, biological set name across every plate being compared.

Note: At least one inter-run calibrator sample must be present in the gene study for inter-run
calibration to occur. Targets without appropriate inter-run calibrator samples will be processed
without correction in the gene study (not recommended).

Inter-run calibrators can be applied in two ways:

n Per target — different PCR primers can have different efficiencies. By default the inter-run
calibrator is applied to all of the wells on the same plate that have the same target name, for
example the Cq generated with the same assay.

n Entire study — one inter-run calibrator is selected by the user and applied to the entire gene
study.

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Chapter 10 Gene Expression Analysis

Gene Study Dialog Box

The Gene Study dialog box includes two tabs:

n Study Setup tab — manages the runs in the gene study.

Important: Adding or removing data files in a gene study does not change the data in the
original file.

n Study Analysis tab — displays the gene expression data for the combined runs.

Study Setup Tab

Table 33 defines the data that appear in the Study Setup tab.

Table 33. Study Setup tab in the Gene Study dialog box

Column Title Description

File Name Name of the run data file (.pcrd extension)

File Folder Directory that stores the data file for each run in the gene study

Date Created Date the run data were collected

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 Gene Study

Table 33. Study Setup tab in the Gene Study dialog box, continued

Column Title Description

Well Group Name Name of the well group that was selected when the file was added to
the gene study
Tip: To analyze one well group in the gene study, you must select
that well group in the Data Analysis window before importing the
data file into the gene study.

Step Protocol step that includes the plate read to collect real-time PCR data

Run Type Either user-defined or PrimePCR™ run

Protocol Edited If selected, indicates that the protocol used for a PrimePCR run was
edited

View Plate Opens a plate map of the plate with the data in each of the runs
included in the Gene Study

Preparing a Gene Study

To prepare a gene study

1. Before importing data into a gene study, do the following in the Data Analysis window:

n Verify that samples containing the same content have the same name. In a gene study,
the software assumes that wells with the same Target or Sample name contain the same
samples.

n Adjust the baseline and threshold (Cq ) in the Quantification tab to optimize the data in
each run.

n Select the well group you want to include in the gene study.

In order to show data from one well group in the gene study, that group must be selected
before importing the data file.

The Study Setup tab shows a list of all the runs in the gene study.

2. In the Gene Study dialog box, choose the Study Setup tab.

3. Click Add Data Files to select a file from a browser window. To quickly add runs to a gene
study, drag the data files (.pcrd extension) into the Study Setup dialog box.

4. CFX Maestro software automatically performs the gene study analysis as you add data files.
Choose the Study Analysis tab to view the results.

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Chapter 10 Gene Expression Analysis

Tor remove runs from the gene study

u Select one or more files in the list and click Remove.

To add notes about the gene study

u Enter notes about the files and analysis in the Notes text box.

Study Analysis Tab

The Study Analysis tab displays the data from all runs in the gene study. The gene expression data
analysis options are the same as those for a single data file with the following exceptions:

n For bar charts, inter-run calibration values (if calculated) appear when you click Inter-run
Calibration.

Note: Only the following sample types can be used as an inter-run calibrator:

o Unknown

o Standard

o Positive Control

Negative control, no template control (NTC), and no reverse transcriptase control (NRT)
sample types cannot be used as an inter-run calibrator.

n For heat maps, if the same targets are in the same location on multiple plates but with different
samples, use the dropdown menu to select a particular plate for analysis.

n The Reference Gene Selection tool identifies the tested reference genes and categorizes
them as Ideal, Acceptable, or Unstable based on their stability:

o Ideal reference genes are stable and represent minimal variations across the tested
samples.

o Acceptable reference genes are not ideally stable and represent moderate variation
across tested samples. Use these reference genes in analysis if no Ideal reference genes
are present.

o Unstable reference genes represent excessive variation across tested samples. It is

recommended that these genes be excluded from analyses.

n The PrimePCR Controls tool displays the results of the tested samples in a table:

o The Summary tab displays a summary of all tested samples. Samples that passed all of
the control assays appear in green. Samples that failed one or more of the control assays
appear in yellow.

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 Gene Study

o The PCR tab displays the results of the positive PCR control assay. This assay detects
inhibition or experimental problems that affect gene expression.

o The RT tab displays the results of the reverse transcription control assay. This assay
qualitatively evaluates RT performance and identifies samples where RT performance is
likely to compromise gene expression.

o The gDNA tab displays the results of the DNA contamination control assay. This assay
determines whether genomic DNA (gDNA) is present in a sample at a level that might
affect qPCR results.

o The RQ tab displays the results of the RNA quality assays (RQ1 and RQ2). These assays
qualitatively assess whether RNA integrity might adversely affect gene expression.

Gene Study Reports

Use the Gene Study Report dialog box to arrange the gene study data into a report. Table 34 lists
all the options available for a gene study report.

Table 34. Categories for a Gene Study report

Category Option Description

Header

Title, subtitle, and logo for the report

Report Information Date, user name, data file name, data file path,
and the selected well group

Gene Study File List List of all the data files in the Gene Study

Notes Notes about the data report

Study Analysis: Bar Chart

Analysis Settings List of the selected analysis parameters

Chart Gene Expression bar chart showing the data

Target Names List of targets in the Gene Study

Sample Names List of samples in the Gene Study

Data Spreadsheet that shows the data

Target Stability Target stability data

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Chapter 10 Gene Expression Analysis

Table 34. Categories for a Gene Study report, continued

Category Option Description

Inter-run Calibration Inter-run calibration data

Box-and-Whisker Chart Gene Expression box-and-whisker chart

Dot-Plot Chart Gene Expression dot plot chart

Study Analysis: Clustergram, Scatter Plot, Volcano Plot, and Heat Map

Analysis Settings Settings for each chart type

Chart Gene Expression chart showing the data

Data Spreadsheet listing the data in each target

Study Analysis: ANOVA Data

ANOVA Settings P-value threshold used in the analysis

ANOVA Results Table of results from ANOVA and Tukey's HSD

post-hoc analysis

Shapiro-Wilk Normality Biological group, count, P-value, and any errors

Test that occur for each target in the analysis

ANOVA Errors Errors identified during ANOVA calculations

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 Gene Study

Creating a Gene Study Report

To create a gene study report

1. Adjust the gene study report data and charts as needed before creating a report.

2. Select Tools > Reports in the Gene Study menu to open the Report dialog box.

3. Choose the options you want to include in the report. The report opens with default options
selected. Select or clear the checkboxes to change whole categories or individual options
within a category.

The section Gene Study Reports that follows lists the available options to display.

4. Change the order of categories and items in a report. Drag the options to the required position.
Items can be reordered only within the categories to which they belong.

5. Click Update Report to update the Report Preview with any changes.

6. Print or save the report. Click the Print Report button in the toolbar to print the current report.
Select File > Save to save the report in PDF (Adobe Acrobat Reader file), MHT (Microsoft
document), or MHTML (Microsoft document) file format and select a location in which to save
the file. Select File > Save As to save the report with a new name or in a new location.

7. (Optional) Create a report template with the information you want. To save the current report
settings in a template, select Template > Save or Save As. Then load the report template the
next time you want to make a new report.

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Appendix A Data Analysis Calculations
CFX Maestro™ software calculates formulas automatically and displays the results in the Data
Analysis tabs. This appendix explains in detail how CFX Maestro software calculates formulas.

P-Value
The P-value is a measure of the statistical significance between the differential expression of a
biological group relative to a control group. It is the probability of obtaining the observed (or more
extreme) result, assuming that there is no difference between the two biological groups.

The P-value is calculated using an unpaired t-test that assumes equal variances between the
groups under analysis. For example, a P-value of 0.05 indicates that there is a 5 percent
probability that this or a more extreme result will randomly occur.

Note: At least two samples are required to determine the P-value for both control and
experimental biological groups. The more samples that are present, the greater the accuracy.
Use of the P-value assumes that the log-converted expression values of the samples within
the biological groups are normally distributed. Use the Shapiro-Wilk Normality Test in the
ANOVA tab to assess normality. For more information, see Shapiro-Wilk Normality Test on
page 219.

The following calculation refers to the P-value displayed within the Graphing and Volcano Plot
tabs. For descriptions of the P-values displayed within the ANOVA tab, see Results Table on page
218.

Note: Expression is converted to a log2 scale before the P-value is calculated.

The formula for P-value is

P-value = 1 – A

Where:

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Appendix A Data Analysis Calculations

Where:

n Γ = gamma function

n t = t-statistic

Where:

n Mean = Arithmetic mean

n NE = Normalized expression

n Count(x) = Size of list x

n SD = Sample standard deviation

Differences in P-Value Results

P-value results might differ depending on the chart you are viewing.

Results Tab

The P-values reported on the Results table are the results of unpaired t-tests comparing the
distributions of per well Normalized Expression (NE) values for the control sample versus the test
sample.

Bar Chart Tab

The P-values reported in the bar chart table are based on the control that is selected on the Bar
Chart tab.

ANOVA Tab

The P-Value ANOVA (uncorrected) and P-Value BH (Benjamini-Hochberg correction for testing
multiple targets) that are reported on the ANOVA table are based on the F statistic from ANOVA,
and indicate whether there are statistically significant differences among the means of the
biological groups for each target. The ANOVA table also shows the results of Tukey's HSD test for
each target. The P-Value Tukey indicates the significance of the difference in means for each
pairwise comparison of biological groups for a given target. The P-Value Tukey is corrected for the
family-wise error rate when testing multiple biological groups.

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 Reaction Efficiency

Shapiro-Wilk Normality Test Tab

The P-value reported in the Shapiro-Wilk test is based on the W-value. In this test, P-value
indicates the probability that the data are obtained from a normally distributed population (null
hypothesis). A significant (small) P-value that rejects the null hypothesis indicates the data are
unlikely to have come from a normal distribution.

Reaction Efficiency
Evidence suggests that using an accurate measure of efficiencies for each primer and probe set
will give you more accurate results when analyzing gene expression data. The default value of
efficiency used in the gene expression calculations is 100%. To evaluate the reaction efficiency,
generate a standard curve using serial dilutions of a representative sample across a relevant
dynamic range and then record the efficiency for subsequent gene expression analysis. If your run
includes a standard curve, then the software automatically calculates the efficiency and displays it
under the Standard Curve on the Quantification tab when Auto Efficiency is checked in the Targets
tab in the Experiment Settings window.

The efficiency (E) in the efficiency formulas refers to the “efficiencies” as described by Pfaffl (2001)
and Vandesompele et al. (2002). In these publications, an efficiency of 2 (perfect doubling with
every cycle) is equivalent to 100% efficiency in this software. You have the option to convert your
efficiency calculations to those used in the software by using the following mathematical
relationships:

n E = (% Efficiency * 0.01) + 1

n % Efficiency = (E – 1) * 100

Relative Quantity
The formula for relative quantity (ΔCq ) for any sample (GOI) is:

Note: This formula is used to calculate Relative Quantity when there is no control sample or
biological group defined.

Where:

n E = Efficiency of primer and probe set. This efficiency is calculated with the formula (%
Efficiency * 0.01) + 1, where 100% efficiency = 2

n C q (min) = Average Cq for the Sample with the lowest average Cq for GOI

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Appendix A Data Analysis Calculations

n C q (sample) = Average Cq for the Sample

n GOI = Gene of interest (one target)

Relative Quantity When a Control Is Selected

When a control sample or biological group is assigned, then the relative quantity (RQ) for any
sample with a gene of interest (GOI) is calculated with this formula:

Where:

n E = Efficiency of primer and probe set. This efficiency is calculated with the formula (%
Efficiency * 0.01) + 1, where 100% efficiency = 2

n Cq (control) = Average Cq for the control sample, or average Cq Mean for the biological group

n Cq (sample) = Average Cq for any samples with a GOI

n GOI = Gene of interest (one target)

Standard Deviation of Relative Quantity

Important: This calculation is applicable only when Analyze Using is set to Samples Only,
Sample Biological Group, or Biological Group Sample.

The formula for standard deviation of the relative quantity is

Where:

n SD Relative Quantity = standard deviation of the relative quantity

n SD CqGOI sample = Standard deviation of the Cq for the sample (GOI)

n Relative Quantity = Relative quantity of the sample

n E = Efficiency of primer and probe set. This efficiency is calculated with the formula (%
Efficiency * 0.01) + 1, where 100% efficiency = 2

n GOI = Gene of interest (one target)

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 Efficiency Corrected Cq (CqE)

Efficiency Corrected Cq (CqE)

The formula for efficiency corrected Cq is

Where:

n E = Efficiency

Mean Efficiency Corrected Cq (MCqE)

The formula for mean efficiency corrected Cq is

Where:

n CqE = Efficiency corrected Cq

n n = Number of replicates

Normalized Expression
Normalized expression (ΔΔCq) is the relative quantity of your target (gene) normalized to the
quantities of the reference targets (genes or sequences) in your biological system. To select
reference targets, open the Experiment Settings window and click the reference column for each
target that serves as a reference gene.

The formula for normalized expression, which uses the calculated Relative Quantity (RQ)
calculation, is

Where:

n RQ = Relative quantity of a sample

n Ref = Reference target in a run that includes one or more reference targets in each sample

n GOI = Gene of interest (one target)

Provided that reference targets do not change their expression level in your biological system, the
calculation of normalized expression will account for loading differences or variations in cell
number that are represented in each of your samples.

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Appendix A Data Analysis Calculations

Expression and Relative Quantity for Biological Groups

When Analyze Using is set to Biological Groups Only, the software displays the average
expression (normalized expression or relative quantity, depending on mode selection) of the
samples within the biological group. Because expression is typically log-normally distributed, the
expression is averaged using the geometric mean:

Where:

n Exp1, Exp2, Expn = Relative quantity or normalized expression of the samples in the biological
group

n n = Number of samples in the biological group

Normalized Expression When a Control Is Selected

When you select a control sample in the Experiment Settings window, the software sets the
expression level of the control sample to 1. In this situation, the software normalizes the relative
quantities of all target (gene) expression to the control quantity (a value of 1). This normalized
expression is equivalent to the unscaled normalized expression analysis when a control is chosen.

Note: This is also known as relative normalized expression (RNE) and fold change.

Standard Deviation for the Normalized Expression

Rescaling the normalized expression value is accomplished by dividing the standard deviation of
the normalized expression by the normalized expression value for the highest or lowest individual
expression levels, depending on the scaling option you choose. The formula for standard deviation
(SD) of the normalization factor is

Where:

n RQ = Relative quantity of a sample

n SD = Standard deviation

n NF = Normalization factor

n Ref = Reference target

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 Normalized Expression Scaled to Highest Expression Level

n n = Number of reference targets

When a control sample is assigned, you do not need to perform this rescaling function on the
standard deviation, as shown in the following formula:

Where:

n NE = Normalized expression

n RQ = Relative quantity of a sample

n SD = Standard deviation

n GOI = Gene of interest (one target)

Normalized Expression Scaled to Highest Expression Level

When the run does not include controls, scale the normalized expression (NE) for each target
(gene) by dividing the expression level of each sample by the highest level of expression in all the
samples. The software sets the highest level of expression to a value of 1 and rescales all the
sample expression levels. The formula for highest scaling is

Where:

n GOI = Gene of interest (target)

Normalized Expression Scaled to Lowest Expression Level

When the run does not include controls, scale the normalized expression (NE) for each target
(gene) by dividing the expression level of each sample by the lowest level of expression in all the
samples. The software sets the lowest level of expression to a value of 1 and rescales all the
sample expression levels. The formula for lowest scaling is

Where:

n GOI = Gene of interest (target)

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Appendix A Data Analysis Calculations

Normalized Expression Scaled to Average Expression Level

When the run does not include controls, scale the normalized expression (NE) for each target
(gene) by dividing the expression level of each sample by the geometric mean level of expression
of all the samples. The software sets the average level of expression to a value of 1 and rescales
all the sample expression levels. The formula for average scaling is

Where:

n GOI = Gene of interest (target)

n GM = Geometric mean of normalized expression for all samples

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 Standard Deviation for the Scaled Normalized Expression

Standard Deviation for the Scaled Normalized Expression

Rescaling the scaled normalized expression (NE) value is accomplished by dividing the standard
deviation (SD) of the normalized expression by the normalized expression value for the highest
(MAX) or lowest (MIN) expression level, depending on which scaling option you choose.

Note: When a control sample is assigned, you do not need to perform this rescaling function
on the standard deviation.

The calculation for this formula is

Where:

n NE = Normalized expression

n SD = Standard deviation

n GOI = Gene of interest (target)

n MAX = Highest expression level

n MIN = Lowest expression level

User Guide | 237

Appendix A Data Analysis Calculations

Error Bars for Standard Deviation(lg) and Standard Error of

the Mean (lg)
In addition to the use of confidence intervals, error bars may be displayed for biological groups
based on the standard deviation or standard error of the mean of the log2 of the expression. The
error bars are calculated as follows:

RQ Lower Error Bar = 2RQ(lg)-SD RQ(lg) or 2RQ(lg)-SEM RQ(lg)

RQ Upper Error Bar = 2RQ(lg)+SD RQ(lg) or 2RQ(lg)+SEM RQ(lg)

Where:

n RQ(lg) = log2 of the relative quantity for the biological group

n SD RQ(lg) = standard deviation of the relative quantity (log2 )

n SEM RQ(lg) = standard error of the mean of the relative quantity (log2 )

Exp. Lower Error Bar = 2Exp.(lg)-SD Exp.(lg) or 2Exp.(lg)-SEM Exp.(lg)

Exp. Upper Error Bar = 2Exp.(lg)+SD Exp.(lg) or 2Exp.(lg)+SEM Exp.(lg)

Where:

n Exp.(lg) = log2 of the expression (normalized expression) for the biological group

n SD RQ(lg) = standard deviation of the expression (log2 )

n SEM RQ(lg) = standard error of the mean of the expression (log2 )

238 | CFX Maestro Software

 Fold Change

Fold Change
Fold change is a measure of the increase or decrease in the expression of a target for an
experimental versus a control sample or biological group and is determined as follows:

If Expression (experimental) > Expression (control):

If Expression (experimental) < Expression (control):

Note: For Graphing, the Expression is based on either the relative quantity or normalized
expression, depending on the selected mode (see Graphing on page 193). However, for the
Scatter Plot, Clustergram, and Heat Map, fold change is always calculated from the
normalized expression.

User Guide | 239

Appendix A Data Analysis Calculations

Corrected Values Formulas

Important: These calculations are applicable only when Analyze Using is set to Samples
Only, Sample Biological Group, or Biological Group Sample.

A difference between corrected values and non-corrected values is seen only if a standard curve is
created as part of the real-time PCR run. The software uses three equations to determine the error
propagation:

n Standard Error

n Standard Error for Normalized Expression

n Standard Error for the Normalized Gene of Interest (target)

The formula for standard error is

Where:

n n = Number of reference targets (genes)

n SD = Standard deviation

The standard error for the normalization factor in the normalized expression formula is

Where:

n n = Number of reference targets

n SE = Standard error

n NF = Normalized expression

n RQ = Relative quantity

The standard error for normalized gene of interest (GOI) formula is

Where:

n SE = Standard error

n GOI = Gene of interest (one target)

240 | CFX Maestro Software

 Confidence Interval Calculation for Biological Group Analysis

n NF = Normalization factor

n n = Number of reference targets

Confidence Interval Calculation for Biological Group

Analysis
When conducting biological groups analysis (Analyze Using is set to Biological Groups Only), the
confidence intervals are calculated for relative quantity and relative normalized expression.
The confidence intervals are calculated in log-scale based on t-distribution using the following
formula:

Where:

n = Mean expression of the log-scale expression levels of the samples in the biological group

n SD = standard deviation of the log-scale expression levels of the samples in the biological
group

n n = number of the samples in the biological group

n t = obtained from the t-distribution based on the degrees of freedom and the alpha level

Note: The alpha level can be set using the P-value threshold field in the Graphing tab.

After the confidence intervals are calculated, they are converted to linear scale and presented in
the Gene Expression Data Table and the bar chart in the Graphing tab.

Box and Whisker Chart Calculations

The Box and Whisker chart displays the distribution of the expression values within a biological
group by plotting the data as quartiles. The 1st and 3rd quartiles are represented by the lower and
upper bounds of the box, respectively. The median is displayed as a solid line across the box. The
whiskers represent the minimum and maximum non-outlier values in the data set. Outliers are
values that exceed the 1st and 3rd quartiles by 1.5 times the inter-quartile range.

Note: If there is only one sample in the biological group it is shown as a single circle,
indicating a single data point.

The following Box and Whisker chart demonstrates how these data is represented.

User Guide | 241

Appendix A Data Analysis Calculations

LEGEND

1. Outlier. This outlier's value is > Q3 + (1.5 x [Q3 - Q1]).

Note: Place the cursor over the circle to view a tooltip that displays the sample name and
the relative quantity or normalized expression information depending on which mode is
selected.

2. Maximum non-outlier demarcation

3. Upper/3rd quartile (Q3). 75% of the expression values are less than Q3.

4. Median, or middle-most value, of the rank-ordered expression values

5. Lower/1st quartile (Q1). 25% of the expression values are less than Q1.

6. Minimum non-outlier demarcation

7. Outlier. This outlier's value is < Q1 - (1.5 x [Q3 - Q1]).

242 | CFX Maestro Software

Appendix B Managing CFX Maestro Users and
Roles
In CFX Maestro™ software, you can create users and assign a role to those users. Roles limit the
access to CFX Maestro features. A user can be assigned only role at a time. However, a CFX
Maestro software administrator can change the user’s role at any time.

Tip: It is not necessary to create users to use CFX Maestro. If you do not create users, all
activity is performed by the default user account admin.

Important: The user admin is the default Administrator account, which you use to initially log
in to CFX Maestro. It is recommended that you create a specific user to administer CFX
Maestro. Assign this user the role of Administrator and perform all administration tasks with
this user.

Managing Users
In the standard edition of CFX Maestro software, user accounts can have any name or password.

To assign a role to each user, select from the list of roles in the User Administration window. In this
example, the Guest user is given the added right to save files.

Adding and Removing Users

Note: Only the CFX Maestro administrator can add and remove users.

To add user accounts to CFX Maestro

1. In the Home window, select User > User Administration.

The User Administration dialog box appears.

User Guide | 243

Appendix B Managing CFX Maestro Users and Roles

2. In the Manage Users pane, type a User Name for the user.

3. Select a user Role.

Roles restrict the rights of the user. The default is Principal.

Tip: You can change the rights for each role. Changing the rights to the role affects all
users who are assigned that role. For more information, see Managing the Rights of Roles
on page 245.

4. (Optional) Type the Full Name and Password for the user.

5. Click OK to open a dialog box and confirm that you want to close the window.

6. Click Yes to close the dialog box and window.

To remove a user

1. In the Manage Users pane, select Remove for each user you want to remove.

2. Click OK to open a dialog box and confirm that you want to close the window.

3. Click Yes to close the dialog box and window.

Note: The list of software users must always include one Administrator.

244 | CFX Maestro Software

 Managing Users

Managing the Rights of Roles

CFX Maestro includes these four roles:

n Administrator (required) — administrators have all rights and you cannot change those rights.
Administrators can also add and remove users and change the rights for each role.

Note: Only an administrator can change the rights for any role.

n Principal — by default the principal user has all rights

n Operator — by default the operator user has all rights except skipping cycles

n Guest — by default the guest user can only read files

Important: Changing rights for a role affects all users assigned that role. You cannot
customize a role for a specific user. Take care when modifying the rights of the roles.

To specify the rights for each role

1. In the Home window, select User > User Administration.

2. In the Manage Rights pane, do either of the following:

n To remove a right from a role, clear its checkbox.

n To add a right to a role, select its checkbox.

3. Click OK to open a dialog box and confirm that you want to close the window.

4. Click Yes to close the dialog box and window.

To reset all rights for all roles

u In the User Administration dialog box, click Restore Default Rights.

User Guide | 245

Appendix B Managing CFX Maestro Users and Roles

Logging In to CFX Maestro Software

CFX Maestro software manages who logs in to the software through the Login dialog box. When
you start the software, CFX Maestro automatically displays the Login dialog box when two or more
users are listed in the User Administration window.

CFX Maestro displays the name of the logged in user at the top of the Home window.

To log in to CFX Maestro

1. In the Login dialog box, select your name from the User Name dropdown list.

2. Type your password.

3. Click OK to close the Login dialog box and open the software.

Changing Users
You can change users while the software is running.

To switch users

1. In the Home window, select User > Select User to open the Login dialog box.

2. Select a name from the User Name dropdown list.

3. Type the new user’s password.

4. Click OK to close the Login dialog box and open the software.

246 | CFX Maestro Software

 Logging In to CFX Maestro Software

Changing User Passwords

CFX Maestro users can change their own password at any time.

To change user passwords

1. In the Home window, select User > Change Password to open the Change Password dialog
box.

2. In Old Password, type your current password.

3. In New Password, type a new password and type it again in Confirm New Password.

4. Click OK to confirm the change.

Viewing Your Role and Rights

Tip: Users assigned the Principal, Operator, or Guest user roles can view only their user
settings, rights, and roles.

To view your current user role and rights

u In the Home window, select User > User Administration.

Contact your CFX Maestro administrator to modify the user settings, rights, and roles listed in the
User Administration window.

User Guide | 247

Appendix B Managing CFX Maestro Users and Roles

248 | CFX Maestro Software

Appendix C LIMS Integration
You can configure CFX Maestro™ software for use with a laboratory information management
system (LIMS). For LIMS integration, CFX Maestro requires plate setup information generated by
the LIMS platform (a LIMS file, *.plrn), a protocol file created using CFX Maestro software (*.prcl), a
defined data export location, and a defined export format.

After the run is finished, CFX Maestro generates a data (.pcrd) file saves it to a defined data export
folder location. CFX Maestro can also create a LIMS-compatible data file in .csv format and save it
to the same location.

Creating LIMS-Compatible Data Files

This appendix explains how to set up CFX Maestro software to create, save, and export
LIMS-compatible data files.

Setting Up LIMS Folder and Data Export Options

By default, CFX Maestro saves the LIMS protocols, files, and data export files to the following
folder:

C:\Users\Public\Documents\Bio-Rad\CFX\LIMS

You can configure CFX Maestro to save the files to another folder, and can change the export
options for LIMS data.

To set up a LIMS folder and data export options

1. On the Home window, select Tools > Options.

2. In the Options dialog box, select Data Export Settings.

User Guide | 249

Appendix C LIMS Integration

3. (Optional) Select Automatically export LIMS data at end of run.

The software will automatically export LIMS data after each run and save it to the specified
location.

4. To change the default export options for LIMS data, click LIMS Data Export Settings.

Important: You can only export LIMS data as a .csv file. Do not change the export format.

5. In the LIMS Data Export Format Settings dialog box, select the required export options and
click OK.

6. In the Options dialog box, navigate to and select a default folder in which you want to save the
LIMS data files. You can select a different location for each file type:

n Protocol

n LIMS file

n Data export

7. Click OK to save the changes and close the Options dialog box.

Creating a LIMS Protocol

To start a LIMS run, create a CFX Maestro protocol file (*.prcl) and save it in the designated LIMS
protocol folder location.

See Chapter 5, Creating Protocols for more information.

250 | CFX Maestro Software

 Creating LIMS-Compatible Data Files

Creating a LIMS File

A LIMS file (*.plrn) contains the plate setup details and the protocol file name. This file is generated
by your internal LIMS. CFX Maestro uses the LIMS file to create a plate file to use with a protocol
file.

CFX Maestro provides two plate import template files that you can edit to create custom LIMS plate
files.

Tip: This task should be performed by a LIMS specialist.

To create a LIMS file

1. In the Home window, select View > Show > LIMS File Folder.

2. Open the LIMS Templates folder and select a .csv file to import into your internal LIMS.

3. Using the LIMS, edit the template file by filling in the required fields listed in Table 35.

4. Save the template with the file name extension .plrn to the LIMS File folder.

Important: CFX Maestro can open only the .plrn file. You must save the .csv file as .plrn in
order to start the LIMS run.

Table 35. Definition of LIMS .csv file contents

Column Row Description Content Purpose

A 1 Plate Header Do not edit Predefined

A,B,C 2 Field/Data/Instruction Do not edit Predefined

B 3 Version Do not edit Predefined

B 4 Plate Size Do not edit Predefined

User Guide | 251

Appendix C LIMS Integration

Table 35. Definition of LIMS .csv file contents, continued

Column Row Description Content Purpose

B 5 Plate Type Enter “BR White,” “BR Required
Clear,” or other
calibrated plate type

B 6 Scan Mode Enter “SYBR/FAM Required

Only:,” “All Channels,”
or “FRET”

B 7 Units Enter one of the Required

following “copy
number,” “fold
dilution,”
“micromoles,”
“nanomoles,”
“picomoles,”
“femtomoles,”
“attomoles,”
“milligrams,”
“micrograms,”
“nanograms,”
“picograms,”
“femtograms,”
“attograms,” or
“percent”

B 8 Run ID Enter short description Optional

or bar code identifying
this run

B 9 Run Note Enter run description Optional

B 10 Run Protocol Enter protocol file Required

name exactly as listed

A 11-15 TBD/Empty Do not edit Predefined

A 16 Plate Data Do not edit Predefined

252 | CFX Maestro Software

 Creating LIMS-Compatible Data Files

Table 35. Definition of LIMS .csv file contents, continued

Column Row Description Content Purpose

A 14-110 Well Position Do not edit Predefined

B-G Ch1 Dye, Ch2 Dye, Enter one calibrated Required

Ch3 Dye, Ch4 Dye, dye name (for
Ch5 Dye, FRET example, “FAM”) for
each channel being
used

H Sample Type Enter one of the Required

following sample types
“Unknown,”
“Standard,” “Positive
Control,” “Negative
Control,” “NTC,” or
“NRT”

I Sample Name Enter sample name Optional

J-O CH1 Target, CH2 Enter target name for Optional

Target, CH3 Target, each channel used
CH4 Target, CH5
Target, FRET Target,

P Biological Set Name Enter biological set Optional

name

Q Replicate Enter a positive Optional

integer for each set of
replicates. The value
cannot be zero

R-W CH1 Quantity, CH2 Enter quantity values Required for all
Quantity, CH3 for any standards. standards
Quantity, CH4 Enter concentration in
Quantity, CH5 decimal form
Quantity, FRET
Quantity

X Well Note Enter well note Optional

User Guide | 253

Appendix C LIMS Integration

Table 35. Definition of LIMS .csv file contents, continued

Column Row Description Content Purpose

Y-AD 14-110 Ch1 Well Color, Ch2 Enter any user-defined Optional
Well Color, Ch3 Well trace style color in a
Color, Ch4 Well 32 bit integer (argb)
Color, Ch5 Well decimal format
Color, FRET Well
Color

Starting a LIMS Run

To start a LIMS run

1. Do one of the following to open a LIMS .plrn file:

n In the Home window, select View > Show > LIMS File Folder and open the target .plrn file.

n In the Home window, select File > Open > LIMS File and open the target .plrn file.

The file opens in the Start Run tab in the Run Setup wizard. The Start Run tab displays
information about the experiment to run. It also displays the connected instrument block or
blocks on which you can run the experiment.

2. In the Start Run tab, select an instrument and click Start Run.

254 | CFX Maestro Software

 Creating LIMS-Compatible Data Files

Exporting Data to a LIMS

When the run completes, CFX Maestro generates a data (.pcrd) file saves it to the defined data
export folder location.

To export the data file to a LIMS

u Open the .pcrd file and select Export > Export to LIMS Folder.

Tip: If you select Automatically Export Data after Run in LIMS Options, CFX Maestro creates a
LIMS-compatible data file in .csv format rand saves it in the same folder.

User Guide | 255

Appendix C LIMS Integration

256 | CFX Maestro Software

Appendix D Troubleshooting CFX Maestro
Software Connection Issues
Application Log
Before starting a new run, the CFX96™ Touch, CFX96 Touch Deep Well™, CFX Connect™, and
CFX384™ Touch instruments initiate a self-diagnostic test to verify that it is running within
specifications. The software records results of this test in the Run Log and Application Log file. If
you notice a problem in one or more experiments, open the run and application logs to find out
when the problem started.

CFX Maestro™ tracks information about the state of an instrument during a run in the Application
Log. Use these logs to track events that occur on instruments and in the software and for
troubleshooting.

To open the Application log

u In the Home window, select View > Application Log.

User Guide | 257

Appendix D Troubleshooting CFX Maestro Software Connection Issues

Troubleshooting
Typically, software and instrument communication problems can be resolved by restarting your
computer and the system. Be sure to save any work in progress before restarting.

Note: Verify that your computer has sufficient RAM and free disk space. The minimum RAM is
4 GB and the minimum hard disk space is 128 GB.

Power Failure
In a power failure, the instrument and computer will shut down. If the power failure is short, the
instrument will resume running a protocol but the Application Log will note the power failure.
Depending on the computer settings and the length of time the power is off, the instrument and
software attempt to continue running depending on the protocol step:

n If the protocol is in a step with no plate read, the protocol continues running as soon as the
instrument regains power.

n If the protocol is in a step with a plate read, the instrument waits for the software to restart and
resume communication to collect the data. In this situation, the protocol continues only if the
software is not shut down by the computer. When the computer and software start up again,
the protocol continues.

Removing Samples from the Reaction Module During Power Outage

You can open a locked motorized lid on a reaction module to remove your samples during a power
failure.

To remove the locking plate

1. Push down on the locking bar to remove the reaction module from the C1000 Touch or CFX
Connect.

2. Carefully set the reaction module on a desk or the lab bench.

3. Position the module so that the front of the module extends 2 inches over the edge.

258 | CFX Maestro Software

 Troubleshooting

4. With an allen wrench, remove the two large screws from under the front edge of the reaction
module (below the button for opening the lid).

You should hear the locking latch release from inside the module.

Important: Do not remove the two small screws located at the front edge of the module.

5. Push the reaction module lid open. Notice that the latch (dark plastic) is no longer attached.
Remove your samples from the block.

6. Replace the locking latch and secure it with the large screws to reassemble the reaction
module with the lid open.

User Guide | 259

Appendix D Troubleshooting CFX Maestro Software Connection Issues

Recovering Data Files to a USB Flash Drive

Note: Only the last 5 run files can be recovered.

To recover data files from stand-alone runs

1. Insert a USB flash drive into the USB port on the thermal cycler base.

2. Tap Tools to open the Tools dialog box.

3. Tap Run Reports.

4. Select the file to recover from the list.

5. Tap Recover Data. The system creates a new data file (.zpcr) and exports it to the USB flash
drive.

Retrieving Files to the CFX Maestro Computer

You can retrieve data and log files located on the instrument, and transfer them to the hard drive of
an attached computer.

Note: All files in the real-time data folder on the instrument base are retrieved to the computer.

To retrieve files from the instrument

1. In the Detected Instruments pane on the Home window, right-click the target instrument and
select one of the following:

n Retrieve Log Files

n Retrieve Data Files

2. Choose a folder location to save the retrieved files.

3. Click Okay.

Installing CFX Maestro Software Manually

To manually install CFX Maestro software

1. If necessary, disconnect any connected instruments from the computer.

Locate and disconnect the instrument’s USB cable on the CFX Maestro computer. The end
inserted in the instrument can remain in place.

2. Log in to the CFX Maestro computer with administrative privileges.

3. Insert the CFX Maestro software USB drive into the computer’s USB port.

4. In Windows Explorer, navigate to and open the CFX Maestro software USB drive.

260 | CFX Maestro Software

 Troubleshooting

5. Open the CFX folder and double-click Setup.exe to install CFX Maestro software.

6. Follow the instructions on the screen to install the software.

When completed, the Bio-Rad CFX Maestro splash screen appears on the computer screen
and the Bio-Rad CFX Maestro icon appears on the desktop.

7. Double-click CFX_Documentation.exe to install CFX Maestro software documentation.

8. After the installation completes, safely eject the software USB drive and start CFX Maestro
software.

Reinstalling the Drivers

To reinstall the instrument drivers

u In the Home window, select Tools > Reinstall Instrument Drivers.

Note: If you have problems with the software communicating with a real-time system after you
reinstall the drivers and check USB connection, contact Bio-Rad Technical Support.

User Guide | 261

Appendix D Troubleshooting CFX Maestro Software Connection Issues

262 | CFX Maestro Software

Appendix E References
1. Sugimoto et al. (1996). Improved thermodynamic parameters and helix initiation factor to
predict stability of DNA duplexes. Nucleic Acids Research 24, 4,501–4,505.

2. Breslauer KJ et al. (1986). Predicting DNA duplex stability from the base sequence. Proc Nat
Acad Sci 83, 3,746–3,750.

3. Hellemans J et al. (2007). qBase relative quantification framework and software for
management and automated analysis of real-time quantitative PCR data. Genome Biol 8, R19.

4. Livak JL et al. (1995). Towards fully automated genome-wide polymorphism screening. Nature
Genetics 9, 341–342.

5. Pfaffl MW (2001). A new mathematical model for relative quantification in real-time RT-PCR.
Nucleic Acids Research 29, 2,002–2,007.

6. Vandesompele J et al. (2002). Accurate normalization of real-time quantitative RT-PCR data

by geometric averaging of multiple internal control genes. Genome Biology 3, 1–12.

7. Fox J (2008). Applied Regression Analysis and Generalized Linear Models. 2nd ed (New
York: SAGE Publications, Inc.).

Minpack Copyright Notice (1999) University of Chicago. All rights reserved

Redistribution and use in source and binary forms, with or without modification, are permitted
provided that the following conditions are met:

1. Redistributions of source code must retain the above copyright notice, this list of conditions,
and the following disclaimer.

2. Redistributions in binary form must reproduce the above copyright notice, this list of
conditions, and the following disclaimer in the documentation and/or other materials provided
with the distribution.

3. The end-user documentation included with the redistribution, if any, must include the following
acknowledgment:

“This product includes software developed by the University of Chicago, as Operator of Argonne
National Laboratory.”

User Guide | 263

Appendix E References

264 | CFX Maestro Software

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Laboratories, Inc.

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