Stratagene QPCR Systems

Stratagene Products
 Stratagene provides a total solution approach to real-time
quantitative PCR (QPCR), simplifying the challenges you face in
sample preparation, assay setup, and data analysis. Whether
you are new to or experienced in QPCR, your individual needs
are met with our comprehensive range of products and industry-
leading support.

Total Real-Time Quantitative PCR Solutions

Stratagene QPCR Instruments and Reagents
from Agilent Technologies

With the addition of Stratagene real-time PCR solutions,

Agilent offers the most comprehensive workflow solution for
gene expression analysis. The Mx3005P system is a flexible
real-time PCR instrument suited for gene expression analy-
sis, validation of microarray data, SNP genotyping, pathogen
detection, DNA methylation assays, and chromatin immuno-
precipitation studies. Brilliant II QPCR reagents offer superior
sensitivity of detection and accommodate rapid cycling for
fast and reliable results.
Discover the Best Tools for Your Specific QPCR Research

PCR Instrument & Reagent Table of Contents

APPLICATION PRODUCT PAGE

QPCR Instruments
Mx3000P and Mx3005P QPCR Systems* 2
High-Performance, Low-Cost QPCR Systems 3
Superior Flexibility and Performance 4
Novel Data Analysis in the Easiest-to-Operate QPCR Software Available 6
QPCR Reagents
Brilliant II QPCR Reagents 8
More Sensitive cDNA Synthesis for Two-Step QRT-PCR Applications 10
RNA Products
QPCR-Grade Reference RNA, DNA-Free 11
Pure RNA from Difficult Samples 12
Selection Guide
Product Ordering List 13
Bioanalysis Technology
Agilent Bioanalyzer and Lab-on-Chip Technology 14

1
The Highest Performance and Flexibility in QPCR Systems
Features of the Mx3000P QPCR Features of the Mx3005P QPCR
System System
The Mx3000P System is a high- The Mx3005P QPCR System expands
performance, full-featured instrument on the Mx3000P system by offering
system designed to accommodate unmatched flexibility and capability to
basic experimental design and offer support even more real-time QPCR
the flexibility required for more applications and chemistries. The
advanced applications (Figure 1). system features five customer-
selected filters and a custom filter
Mx3000P QPCR System path feature to support detection of a
• Four optical channels with user- broad range of dyes (including FRET-
selected filters for greater flexibility based fluorescence and large stoke
• Broad wavelength range excitation shifts). The enhanced features of the
supports most fluorescent dyes Mx3005P system will accommodate
• Single photomultiplier tube (PMT) your research needs now and in the
for detection ensures superior future (Figure 1 and 2).
sensitivity, reproducibility, and
linear dynamic range to 10 orders of Mx3005P QPCR System
magnitude
• Five optical channels with user-
selected filters for greater
flexibility

• Defined excitation and emission

detection wavelengths are ideal for
superior multiplex results, up to five
targets simultaneously (Figure 7)

• Open platform design supports

virtually all fluorescent chemistries
and numerous applications

Figure 1. Figure 2.
The Mx3000P QPCR System The Mx3005P QPCR System
Four-color, 96-well format full-featured QPCR system. Five-color, 96-well format, open platform QPCR
[Also includes MxPro QPCR Software) system with independent filter wheel control for
greater dye flexibility. (Also includes MxPro QPCR
Software)
2
 The First High-Performance, Low-Cost QPCR System
Affordable QPCR, Without Sacrificing Coming to You in Real-Time A
Performance
The Mx3000P QPCR System brings • Four optical channels with user-
real-time QPCR instrumentation to the selected filters for greater flexibility
individual researcher with a limited
budget. The precision optical design • Easy to use MxPro QPCR Software
coupled with highly uniform thermal for simple assay setup and data
performance generates consistent analysis
results in even the most challenging
applications (Figure 3 and 4). • Narrow wavelength excitation and
emission detection wavelengths
minimizes signal contamination and
ensures reproducible results
B
• Single photomultiplier tube for
detection ensures superior
sensitivity, reproducibility, and
linear dynamic range to ten orders
of magnitude

Figure 4.
Ten Orders of Magnitude Dynamic Range
The low signal to noise performance of the
photomultiplier tube supports detection across a
large copy number range. (A) Eight replicates of
ten fold dilutions of plasmid DNA for ß-actin target.
(B) Standard curve displayed with 99% confidence
intervals and efficiency=98% and Rsq=0.999.

Figure 3.
Two-Fold Dilution Series
The superior optical system supports accurate
quantification of two-fold differences in low copy
number down to less than 10 copies. Four replicates
of a dilution series from 20,000 to 1.2 copy equivalent
of plasmid containing ß-actin target detected with a
molecular beacon. Average delta Ct between dilutions
is 0.95 cycles.

3
Superior Flexibility and High-Performance
The Mx3005P Real-Time PCR System advances the proven Mx3000P System. Offering unmatched
flexibility and capability, the Mx3005P System can support even more real-time QPCR
applications and chemistries to accommodate your research needs now and in the future.

Advanced Optical System photomultiplier tube (PMT) has a

The Mx3005P System features the large dynamic range of detection and
same optical scanning design as the a low signal to noise ratio, allowing
Mx3000P system. A scanning fiber low- to high-abundance targets to be
optic head ensures all wells: accurately quantified. Excitation light
is generated by a halogen lamp and
1) receive the same amount of delivered to the sample through fiber
excitation light, optics. The fiber optic bundle is coaxial
so it delivers excitation light and
2) are detected for the same amount simultaneously detects fluorescence
of time, and emission from the sample. Detected
light is delivered to the PMT via the
3) are the same distance from the fiber optic cable (Figure 5).
detector.
The precision optic scanning design
The scanning motion system of the with a single light source and single
fiber optic cable eliminates optical detector provides uniform excitation
variation based on well position in and detection coupled with a uniform
the 96-well block, thus eliminating thermal system ensures highly
the need for signal correction by reproducible results (Figure 6).
calibration or reference dyes. The

Figure 5 Figure 6.
Optical System Design 96-Well Uniformity
The halogen lamp in the instrument systems provides a wide-range of excitation A precision thermal design with high-performance optics supports excellent
allowing more dye flexibility with more intensity than standard light emitting well-to-well uniformity. SYBR® Green I uniformity assay for ß-actin containing
diodes (LEDs). The excitation and emission filters are defined to narrow plasmid. Average Ct value at threshold is 18.1 and standard deviation of Ct values
is 0.05. Ct range across 96 wells is 0.26 cycles (18.00 to 18.26).
wavelengths to minimize fluorescence signal crosstalk. Fiber optic bundles
channel the light into the plate and back to the PMT to ensure minimal signal loss.

4
 Powerful Data Analysis Software
Mx3005P Optical Filters Powerful Data Analysis Software
The new five-position user The Mx3005P system uses the most
customizable filter wheel design offers advanced version of our graphical user
a multitude of dye choices, multiplex interface and data analysis software.
dye combinations enabling up to five The MxPro QPCR Software is easy to
targets per well, and the ability to use and organized by application so
create custom excitation and emission you can easily navigate the software
filter pairs that can accommodate the and run assays quickly.
rapidly expanding list of fluorescence
dyes. You can choose which five Optical Sytem Features
filters are installed in the instrument
from a list of eight filter sets spanning • Five optical channels with user-
deep blue dyes to far red dyes thus selected filters for greater flexibility
maximizing the useable wavelength
spectrum (Table 7). • Defined excitation and emission
detection wavelengths are ideal
Precision Thermal System for superior multiplex results, up to
The thermal system of the Mx3005P five fluorescent dyes simultaneously
system shares the same Peltier-based (Figure 7)
design with the Mx3000P system. This
system ensures uniform ramping and • Custom filter path selection to
thermal accuracy for amplification support matching of different
reproducibility from well-to-well and excitation and emission filters
run-to-run (Figure 8).
• Flexible platform design supports
virtually all fluorescent chemistries
and numerous applications

Figure 7. Figure 8
5-Plex Standard Curves RNA Quantification by RiboGreen® Plate Read Experiment
Five target multiplex assay on the Mx3005P system using Alexa Fluor® 350, Our MxPro Software supports plate read applications for quantifying nucleic acid
FAM™, HEX™, ROX™, and Cy™5 filters. Three replicates of 4-fold dilutions of QPCR preparations before running QPCR. Three replicates of RNA standards from
Human Universal Reference cDNA detecting five gene targets simultaneously. 500 pg to 5 ng. Standard curve generated from plate read experiment
Detection from the highest abundance to the lowest abundance gene target automatically quantitates RNA concentration in unknown samples. Ideal for
(CYCLO to ENOS gene targets) spans a Ct range of 17-37 (delta Ct = 20). quick and accurate quantification of RNA samples before performing gene
expression QPCR experiments.

5
Novel Data Analysis in the Easiest-to-Operate QPCR
Software Available
The MxPro QPCR Software for the Mx3000P and Mx3005P Systems combines cutting-edge data
analysis algorithms with intuitive organization designed for ultimate ease-of-use.

Setting Up and Running the

MxPro QPCR Software Features Experiment
The experimental setup features well
• Multiple Experiment Analysis definition by target assay name (e.g.,
functionality FAM = p53), automated standard
curve setup, customized well naming,
• One click setup of plate layout and importing plates, and a flexible thermal
thermal profile for quick start runs profile setup (Figure 9).

• View and analyze data in real-time Data Analysis and Reporting the
Results
• Multiple customizable data The data analysis module includes two
analysis algorithms for baseline automated and customizable methods
�

MxPro correction and threshold setting for baseline subtraction and threshold
setting. The “Adaptive Baseline”
• Export images directly from the algorithm dynamically assigns baseline
software, export the raw data to start and end cycles independently for
re-create the image, and export the all amplification plots (Figure 10).
text data in multiple formats

• 21 CFR Part 11 support

Figure 9. Figure 10.

Flexible, Easy to Use Plate Setup Multiple Data Analysis Views
Import Plate Setup from previous experiments or templates. Assign assay/gene The Adaptive Baseline algorithm ensures accurate quantification across all
target names to dyes. Setup standard curve and replicates using auto-increment five gene targets. Amplification plot and standard curve data can be displayed
feature. Add well information to identify sample name, number, quantity, or any simultaneously and changes in threshold are instantly displayed on the standard
curve.
other sample specific information.

6
Generating Data in Multiple Formats
The Comparative Quantification 8

module automatically determines gene

7

expression fold change, calculates

5

statistical error, and generates a 3

Log fold Change to Cal.(Log base 2) (dRn)

2

publication quality chart (Figure 11). 1

After analysis, the MxPro QPCR

0

-1

software is capable of creating custom -2

-3

reports and exporting all plots, charts, -4

and labels (Figure 12). Images can

-5

-6

be directly exported to Microsoft®

-7

-8
36-p53 37-p53-1 38-p21 39-p21-1 40-Fas 41-Fas-1 42-FasL 43-FasL1 44-Bax-1 45-Bax-1 46-Bcl 47-Bc1-1

PowerPoint®, and high resolution Replicate/Well ID

bitmap images, chart and plot data Figure 11.

can be exported to Microsoft® Excel®, Automated Analysis of Gene Expression Data
The Comparative Quantitation module in MxPro Software automatically
using .xml, and .txt formats to easily calculates relative quantity for gene expression experiments. Data is
re-create the data in different formats displayed as normalized fold gene expression to a reference control on a
(Figure 13). All text data can also be log(2) scale with upper and lower error limits based on variation in the
replicates. In the figure above, fold expression change for six genes across
exported in any of these formats. two treatments is displayed.

Multiple Experiment Analysis

New Multiple Experiment Analysis
allows users to analyze up to 12
separate plates in one project. The
power and convenience of this will
enable users to speed through their
work— without repetitive and time-
consuming procedures.

21 CFR Part 11 Compatible

The 21 CFR Part 11 Compatible
features activates secure application
login, database file management,
and data file audit trails. The audit
trail will record changes made to the
Figure 12.
data which affect the final result, and Create Custom Data Reports
reports can be generated for the audit Create a custom data report by determining report format and which
trail, user accounts, and error logs. data sets to display. In the figure above, Plate Setup, Thermal Profile,
Amplification Plots, Standard Curve, and Text Report are selected for the
report.

å Export to
Excel®

Figure 13.
MxPro QPCR Software Data Exports
å Export to
PowerPoint®

The MxPro Software is designed with the flexibility to export data in many formats, including data images, charts, and text exported in multiple formats. In the figure
above, ten orders of dynamic range amplification plot and standard curve data are displayed in the MxPro Software, then exported in one click to Microsoft® PowerPoint®
and Excel®.

7
Brilliant II Real-Time Quantitative PCR Reagents
Superior Sensitivity for Improved Sensitive Detection of DNA or RNA
Quantification and Reproducibility using SYBR Green Dye
Our next generation Brilliant II QPCR Our Brilliant II SYBR Green QPCR
and QRT-PCR Master Mix Kits offer Master Mix exhibits earlier Ct
superior sensitivity for improved detection with over twice the
quantification and reproducibility. fluorescent intensity compared to
our original Brilliant SYBR Green
A
The New Brilliant II Master Mix Kits product and higher fluorescence
Feature: than many competitor reagents. We
also optimized cycling conditions for
• Improved performance with more rapid run times with increased
Fluorescence (dRN)

0.1
earlier Ct detection across a wide performance when compared to other
2.5 copy
5 copy
dynamic range commercially available products that
recommend longer duration cycling
• New formulations available with conditions.
0.01
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 low and high concentrations of
Cycles
ROX reference dye Sensitive and Specific Probe-Based
Detection
B 38 Log fit values • Improved data reproducibility The sensitivity of Brilliant II reagents
Bril2 Standards, RSq:0.998
37
36
Bril2, Y= -3.451*LOG(X) + 38.38. Eff.= 94.9% • Greater flexibility for use with also improves replicate reproducibility
35 numerous different templates and at lower concentrations and overall
targets
34
reaction efficiency across the entire
Ct (dRn)

33
32
31
range of concentration. We also tested
30 • More accurate quantification the ability to reproducibly distinguish
29
28
results small differences in template
27
10 100 1000
concentrations. Our Brilliant II QPCR
Initial Quantity (relative) • Increased sensitivity of detection reagents are capable of quantifying
to low copy numbers 2-fold differences (equal to 1 cycle or
Figure 14
Superior Sensitivity and Precision to Detect Target 1 Ct difference) in samples between
Across 2-Fold Dilutions 5 and 2.5 copy equivalents at 95%
Amplification plot (semi-log) of 2-fold dilutions of efficiency (see Figure 14).
linearized plasmid (14A). Brilliant II QPCR reagents
exhibit precise detection of 2-fold differences from
1280 copies down to ~2.5 copies. The average delta Using the table below and on page 10
Ct across all 10 dilutions is 1 cycle and the standard you can select the appropriate reagent
curve efficiency equals 95% (14B).
kit for your real-time PCR instrument.

Format DNA (cDNA) Quantification RNA Quantification 2-Step

Master Mix Brilliant II QPCR Master Mix Brilliant II QRT-PCR Master Mix, Brilliant II QRT-PCR,
(Cat No. 600804) 1-Step AffinityScript Master Mix,
(Cat No. 600809) 2-Step
Brilliant II FAST QPCR Master Mix
(Cat No. 600827)
(Cat No. 600845)
Master Mix with ROX Brilliant II QPCR Low ROX Master Brilliant II QPCR Low ROX Master AffinityScript QPCR cDNA
Reference Dye Premixed Mix (Cat No. 600806) Mix, 1-Step (Cat No. 600837) Synthesis Kit (Cat No. 600559)
(Low and High ROX plus
concentatrations for Brilliant II QPCR High ROX Master Brilliant II QPCR High ROX Master Brilliant II QPCR Low ROX
different QPCR systems) Mix (Cat No. 600805) Mix, 1-Step (Cat No. 600838) Master Mix (Cat No. 600806)
or
Brilliant II QPCR High ROX
Master Mix (Cat No. 600805)
RNA Preparation and SideStep II QRT-PCR Master Mix SideStep II QRT-PCR Master
Amplification Kit,1-Step (Cat No. 400917) Mix Kit 2-Step (Cat No. 400918)

Table 1.
Select the appropriate probe-based detection kit for your real-time PCR instrument

8
 Brilliant II FAST QPCR Reagents
Designed to Perform • Simultaneously amplifies low and
Introducing the Next Generation of high abundance targets
Brilliant II FAST Master Mixes. These • Maximizes analysis of limited or
reagents were developed to create a rare samples
system for completing QPCR reagents
in less time without compromising • Allows detection of multiple
target detection sensitivity, specificity targets plus an internal control
or reproducibility.
• Saves time and increases
• Accelerated cycling protocol that throughput
completes 40 cycles of PCR in
approximately 48 minutes Alien Reference RNA QRT-PCR
Detection Kit
• Rapid Hotstart capability to The new Alien reference RNA is
minimize non-specific product a valuable tool for validating the
formation while reducing run time quality of experimental RNA samples,
interpreting the quality of QRT-PCR
• Improved performance with data, and monitoring the overall
earlier Ct detection across a wide performance of QRT-PCR assay
Company A, RSq:0.999
34 Company A, Y = -3.573*LOG(X) + 23.39, Eff. = 90.5%
33 Company I, RSq:0.996

dynamic range reagents and instrumentation.

32 Company I, Y = -3.387*LOG(X) + 23.34, Eff. = 97.4%
31
30 Company Q, RSq:0.998
29 Company Q, Y = -3.443*LOG(X) + 22.79, Eff. = 95.2%
28 Brilliant® II, RSq:0.998
27 Stratagene, Y = -3.316*LOG(X) + 21.59, Eff. = 100.2%
26
25

• Increased sensitivity of detection • Non-homologous to known

24

Ct (dR)
23
22

to low copy numbers sequences

21
20
19
18
17
16
15
14

Multiplex Up to Four Targets • High-quality external control

13
12
0.001 0.01 0.1 1 10 100 1000

Simultaneously
Initial Quantity (ng)

Figure 15.
The Brilliant Multiplex QPCR Master • Use of Probe-Based Detection Brilliant II QPCR Reagents Deliver Better Sensitivity
Mix is ideal for QPCR amplification enables gene of interest and Alien and Reproducibility Over a Wide Range of Concentra-
tions
of three to five targets in a single RNA to be run in the same tube Standard curve plot of 10-fold dilutions in duplicate
reaction (Figure 15). using multiplex detection from 300 ng down to 30 pg of cDNA. The Brilliant II
master mix detects template at ~1-2 Cts earlier on
average, with better efficiency, and tighter replicates
• Amplifies up to 4 targets in a • Ideally suited for assay at the lower concentrations (signal detection by a
single reaction standardization applications linear hydrolysis probe for cyclophillin target).

Format DNA (cDNA) Quantification RNA Quantification 2-Step

Master Mix Brilliant II SYBR Green QPCR
®
Brilliant II SYBR Green
®
Brilliant II SYBR® Green
Master Mix (Cat No. 600828) QRT-PCR Master Mix,1-Step QRT-PCR AffinityScript
(Cat No. 600825) Master Mix,
Brilliant II FAST SYBR® QPCR 2-Step (Cat No. 600834)
Master Mix
(Cat No. 600843)
Master Mix with ROX Brilliant II SYBR® Green QPCR Low Brilliant II QPCR Low ROX AffinityScript QPCR cDNA
Reference Dye Premixed ROX Master Mix (Cat No. 600830) Master Mix, 1-Step (Cat No. Synthesis Kit (Cat No. 600559)
(Low and High ROX 600835) plus
concentatrations for different Brilliant II SYBR® Green QPCR High Brilliant II QPCR High ROX Brilliant II SYBR® QPCR Low ROX
QPCR systems) ROX Master Mix (Cat No. 600829) Master Mix, 1-Step (Cat No. Master Mix (Cat No. 600830)
600836) or
Brilliant II SYBR® QPCR High
ROX Master Mix (Cat No.
600829)
RNA/DNA Preparation and Brilliant SYBR® Green SideStep SideStep II SYBR® Green
Amplification QPCR Master Mix QRT-PCR Master Mix Kit
(Cat No. 400904) 2-Step (Cat No. 400909)

Table 2.
Select the appropriate SYBR-based detection kit for your real-time PCR instrument

9
More Sensitive cDNA Synthesis for Two-Step QRT- PCR
Applications
Following RNA purification, or cell lysate generation, one-step or two-step QRT-PCR is employed. For
the analysis of multiple genes from a single sample, RNA is converted into cDNA. An aliquot of cDNA
can then be removed and PCR-amplified in real time. Our cDNA synthesis and two-step QRT-PCR kits
are exclusively master mix formats for highest reproducibility and sensitivity. Importantly, all are
qualified for use in real-time applications.

cDNA Synthesis for QPCR Robust Reverse Transcription

The AffinityScript QPCR cDNA Our AffinityScript multiple temperature
Synthesis Kita is designed for high- reverse transcriptase synthesizes
efficiency conversion of RNA to cDNA a complementary DNA strand from
and is fully optimized for real-time single-stranded RNA, DNA, or an RNA:
quantitative PCR applications (Figure DNA hybrid.
16). The high sensitivity obtained with
this kit is due to the inclusion of our Two-Step QRT-PCR Kits Using Affinity
QPCR-grade AffinityScript Multiple Script QPCR cDNA Synthesis Kit
Temperature Reverse Transcriptase Achieve sensitive and specific QRT-
(RT) and a high-efficiency cDNA PCR in a flexible two-step format.
synthesis buffer. The AffinityScript These kits are composed of two
QPCR cDNA synthesis kit is formatted modules. First, RNA is reverse-
as a master mix that reduces pipetting transcribed using the AffinityScript
steps and enhances reproducibility QPCR cDNA synthesis kit, using our
in two-step QRT-PCR. It also employs QPCR-grade, AffinityScript RT in just
a fast, 15-minute protocol for most 15 minutes. The cDNA of interest
targets. is then quantified using the second
real-time PCR module, using any of
our Brilliant II SYBR or probe-based
master mixes.

Temperature (°C) RNA Amount Ct (dRn)

1.3
42 100 ng 19.27
1.2
1.1 42 10 23.00
1.0 42 1 26.59
100 ng
Fluorescence (dRN)

0.9 42 NTC No Ct
0.8
10 ng 50 100 ng 19.30
0.7
0.6 50 10 22.83
0.5 1 ng 50 1 26.31
0.4 50 NTC No Ct
0.3 55 100 ng 19.44
0.2
0.1 NTC 55 10 23.12
0.0 55 1 26.92
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
55 NTC No Ct
Cycles
Figure 16.
The AffinityScript QPCR cDNA Synthesis Kit Delivers Robust cDNA Synthesis Across Multiple Temperatures
We synthesized cDNA from total RNA using different reaction temperatures (42, 50, and 55°C) with oligo dT primers. Template amounts indicated are RNA amounts added
at the cDNA synthesis level (100 ng, 10 ng, 1 ng). Using our Brilliant SYBR® Green QPCR Master Mix for QPCR amplification of the ODC gene, the same Ct was observed for
each RNA input regardless of RT reaction temperature (Panel A). For example, using 100 ng, the Ct for 42°C was 19.27; for 50°C, 19.30; for 55°C, 19.44 (Panel B).

10
 QPCR-Grade Reference RNA, DNA-Free

Stratagene QPCR Reference Total RNAs are high-quality, universal RNA controls for quantitative PCR
gene-expression analysis. In order to reliably compare data across multiple experiments and
instruments, it is essential to have a constant reference material to assess the performance of each
QPCR reaction and quantify gene expression levels.

Maximal Gene Representation We offer QPCR reference total RNA

The Stratagene QPCR Reference Total from both human and mouse cells.
RNAs are used to directly compare The QPCR Human Reference Total
data between multiple experiments RNA is a collection of RNA pooled
(Figure 17). These RNAs are extracted from 10 human cell lines derived from
from quality cell lines representing different tissues. The QPCR Mouse
different human and mouse tissues, Reference Total RNA is derived from
pooled together for maximal gene RNA pooled from 11 mouse cell lines
representation. The broad gene which are also derived from tissues.
coverage allows you to use the RNA We choose cell lines, rather than
reference material as a universal tissues, as our starting material since
reference for nearly any human or we have found that this is the most
mouse gene being investigated in consistent and highest quality source
QPCR experiments. These high-quality of RNA.
human or mouse total RNA controls
are manufactured for maximum Stratagene QPCR Human Refereence RNA (QHRR)
representation of low, medium, and
high-abundance gene transcripts. Experiment #1 Experiment #2
Our QPCR reference total RNAs are QHRR
Primers and/or Probes Primers and/or Probes
qualified for QPCR and guaranteed (Gene of Interest + Endogenous Control) (Gene of Interest + Endogenous Control)
to be free of genomic DNA. We
manufacture these control RNAs at Primers
and/or
industrial scale lot sizes and perform Probes
(Endogenous
stringent quality control to maximize Control)

lot-to-lot reproducibility.

Treated Untreated QHRR QHRR Treated Untreated

Sample Sample Sample Sample

Real-Time QRT-PCR Real-Time QRT-PCR

gene
M Stratag
Stratagene
Mx3000P and Mx3005P
Real-Time PCR Systems

Directly Compare Data

Normalize Samples to QHRR Normalize Samples to QHRR
Directly Compare Data
Figure 17.
Accurately Measure Relative Expression Levels and Compare Data from Multiple Experiments
Use the Stratagene QPCR Reference Total RNA to measure endogenous control levels alongside your treated and
untreated samples. Normalize samples to the endogenous control to determine relative expression levels of your
gene of interest.

11
Pure RNA from Difficult Samples
Archived tissue specimens are rich sources of material for extensive analysis of mRNA expression
utilizing QRT-PCR. However, the chemical nature of fixatives and fixation time significantly affects the
quality of RNA and DNA extracted from these tissues. Moreover, reliable recovery of RNA from small
cell populations, such as LMD cells, are also particularly challenging to analyze by QRT-PCR.

Formalin-Fixed Paraffin-Embedded modifications and therefore more RNA

(FFPE) Tissue is accessible for real-time QRT-PCR
The Absolutely RNA FFPE Kit delivers analysis than with other kits. High,
a reliable, easy to use, non-toxic medium, and low abundance genes
method to isolate quality RNA from can be quantified reliably and
difficult paraffin-embedded tissue calibrated against the QPCR-grade
sections and includes a quantitative reference RNA (Figure 18).
total RNA reference standard for
meaningful QRT-PCR expression Laser-Microdissected Cells
analysis. Using a standard proteinase Laser micro-dissection (LMD) is a
K digestion protocol is ineffective technique used to isolate a pure
at reversing the RNA modifications subpopulation of cells from tissue
caused by the fixation process, sections containing a heterogeneous
resulting in poor and unreliable RNA mixture for detailed molecular
recovery. In contrast, our method analysis. Isolating high-quality total
completely reverses the RNA RNA from LMD-recovered cells can be
challenging and inefficient when
standard methods are used. In
contrast, our Absolutely RNA
Microprep and Nanoprep Kits
effectively isolate pure RNA from
small samples while avoiding ethanol
precipitation. The Absolutely RNA
nanoprep kit is the only kit qualified
to isolate RNA from a single cell. It
elutes pure RNA into small, 10-µl
elution volumes using our specially
designed spin cups (Table 3).

Figure 18.
The Absolutely RNA FFPE Kit Extracts RNA of Sufficient Yield and Concentration
for Downstream QRT-PCR Analysis
Cyclophilin gene expression level was determined using RNA isolated from
various formalin fixed paraffin-embedded (FFPE) human tissues using the
Absolutely RNA FFPE Kit. The Stratagene QPCR Total Human Reference RNA was
used to calibrate expression levels. (Mx3000P QPCR System)

Reagent Absolutely RNA Miniprep Kit Absolutely RNA Absolutely RNA 96 Absolutely RNA Nanaprep Kit Absolutely RNA FFPE Kit
Microprep Kit Microprep Kit
Sample Size 105 to 107 cells or 5-40 mg of Up to 5 x 105 cells Up to 5 x 105 cells 1 x 104 cells down to a single cell Formalin-fixed paraffin
tissue embedded tissues

Format Fiber matrix spin cup Fiber matrix spin cup Fiber matrix spin cup 96- Fiber matrix spin cup Fiber matrix spin cup
well format

Capacity > 200 μg RNA 50 μg RNA 50 μg RNA Approx. 200 ng RNA 50 μg RNA
Elution Volume 50 μl 30 μl 30 μl 10 μl 30 μl

Table 3
Choose the Absolutely RNA Kit that Suits Your Sample Type and Size

12
 Product Listings
QPCR Systems Catalog No.
Mx3000P QPCR System 4-color system (110v) with notebook computer 401403
4-color system (110v) with desktop computer 401405
4-color system (230v) with notebook computer 401406
4-color system (230v) with desktop computer 401407
Mx3005P QPCR System 5-color system (110v) with notebook computer 401449
5-color system (110v) with desktop computer 401456
5-color system (230v) with notebook computer 401457
5-color system (230v) with desktop computer 401458
MxPro ET (Electronic Tracking) QPCR Software
New and existing Mx QPCR System users 401467
Multi-user license agreement for six users 401468
QPCR and QRT-PCR Reagents
Brilliant II QPCR Master Mix 400 rxn 600804
Brilliant II QPCR Master Mix (10 pack) 4000 rxn 600815
Brilliant II QRT-PCR Master Mix, 1-Step 400 rxn 600809
Brilliant II QRT-PCR Master Mix, 1-Step (10 pack) 4000 rxn 600818
Brilliant II QRT-PCR Core Reagent Kit, 1-Step 400 rxn 600810
Brilliant II QRT-PCR Core Reagent Kit, 1-Step (10 pack) 4000 rxn 600819
Brilliant II QRT-PCR, AffinityScript Master Mix, 2-step 400 rxn 600827
Brilliant II QPCR Low ROX Master Mix 400 rxn 600806
Brilliant II QPCR Low ROX Master Mix (10 pack) 4000 rxn 600817
Brilliant II QPCR High ROX Master Mix 400 rxn 600805
Brilliant II QPCR High ROX Master Mix (10 pack) 4000 rxn 600816
Brilliant II QRT-PCR Low ROX Master Mix, 1-Step 400 rxn 600837
Brilliant II QRT-PCR Low ROX Master Mix, 1-Step (10 pack) 4000 rxn 600841
Brilliant II QRT-PCR High ROX Master Mix, 1-Step 400 rxn 600838
Brilliant II QRT-PCR High ROX Master Mix, 1-Step (10 pack) 4000 rxn 600842
Brilliant II SYBR Green QPCR Master Mix 400 rxn 600828
Brilliant II SYBR Green QPCR Master Mix (10 pack) 4000 rxn 600831
Brilliant II SYBR Green Low Rox Master Mix 400 rxn 600830
Brilliant II SYBR Green High Rox Master Mix 400 rxn 600829
Brilliant II SYBR Green High Rox Master Mix (10 pack) 4000 rxn 600832
Brilliant II SYBR Green Low Rox Master Mix (10 pack) 4000 rxn 600833
Brilliant II SYBR Green QRT-PCR Master Mix, 1-Step 400 rxn 600825
Brilliant II SYBR Green QRT-PCR Master Mix, 1-Step 4000 rxn 600826
Brilliant II SYBR Green QRT-PCR AffinityScript Master Mix, 2-step 400 rxn 600834
Brilliant II SYBR QRT-PCR Low ROX Master Mix, 1-Step 400 rxn 600835
Brilliant II SYBR QRT-PCR High ROX Master Mix, 1-Step 4000 rxn 600836
Brilliant II SYBR QRT-PCR Low ROX Master Mix, 1-Step (10 pack) 4000 rxn 600839
Brilliant II SYBR QRT-PCR High ROX Master Mix, 1-Step (10 pack) 4000 rxn 600840
Brilliant II FAST SYBR Green QPCR Master Mix 400 rxn 600843
Brilliant II FAST SYBR Green QPCR Master Mix (10 pack) 4000 rxn 600844
Brilliant II FAST QPCR Master Mix 400 rxn 600845
Brilliant II FAST QPCR Master Mix (10 pack) 4000 rxn 600845
Sample Preparation
Absolutely RNA FFPE Kit 50 preps 400809
Absolutely RNA FFPE Kit (w/o deparaffinization reagents) 50 preps 400811
Highly Pure Total RNA
Absolutely RNA Microprep Kit 50 preps 400805
Absolutely RNA 96 Microprep Kit 2 plates 400793
10 plates 400794
Absolutely RNA Miniprep Kit 50 preps 400800
Absolutely RNA Nanoprep Kit 50 preps 400753
Messenger RNA Isolation
Absolutely mRNA Purification Kit 10 preps 400806
cDNA Synthesis for Two-Step QRT-PCR
AffinityScript Multiple Temperature Reverse Transcriptase 10 rxn 600105
50 rxn 600107
200 rxn 600109
Universal QPCR Control RNAs
AffinityScript QPCR cDNA Synthesis Kit 50 rxn 600559
Stratagene QPCR Reference Total RNA, Human 25 µg 750500
Stratagene QPCR Reference Total RNA, Mouse 25 µg 750600

13
Sophisticated Bioanalysis Instrumentation
The Agilent 2100 Bioanalyzer Applied Lab-on-a-Chip Technology
• A complete array of applications The Agilent 2100 Bioanalyzer is
(DNA, RNA, proteins, and cells) an indispensible tool for molecular
biology labs. It allows the objective
• The gold standard for RNA QC, measurement of RNA samples using
21CFR Part 11 compliance, and our patented RIN algorithm,
full-compliance services size, and concentration determination
of DNA or protein samples, and
• Sophisticated user-friendly measurement of unique cellular events
software with multiple functionality using the cell application. Utilizing
Lab-on-a-Chip technology, the Agilent
2100 Bioanalyzer combines multiple
sample analysis procedures (sample
handling, separation, staining, and
detection) on a single chip, thereby
improving precision and reproducibility
over traditional electrophoretic
methods. A full line of validated
reagents ensures consistent, reliable
results.

Agilent Bioanalyzer Instruments Catalog No.

2100 Electrophoresis Bioanalyzer (PC not included) G2939AA

2100 Bioanalyzer Desktop Bundle (cartridge not included) G2940CA
2100 Bioanalyzer Electrophoresis Set (cartridge/license) G2947CA
2100 Bioanalyzer Flow Cytometry Set (cartridge/license) G2948CA
2100 Bioanalyzer Laptop Bundle (cartridge not included) G2943CA
2100 Bioanalyzer Electrophoresis Set (cartridge/license) G2947CA
2100 Bioanalyzer Flow Cytometry Set (cartridge/license) G2948CA
Desktop Bundle (computer & SW upgrade only) G2950CA
Laptop Bundle (computer & SW upgrade only) G2953CA

14
 Gold Standard Lab-on-a-Chip Technology

RNA Protein

DNA Cell

RNA Solutions Description Contents Catalog No.

RNA 6000 Nano Kits For analysis and quantitation of messenger and total RNA in the Reagents, supplies, 25 chips, 2 cleaning chips, 5067-1511
low- to mid- nanogram range 1 syringe, and reagent kit guide. A lower marker and
ladder is included with the RNA Nano reagents
RNA 6000 Pico Kits For analysis and quantitation of messenger and total RNA in the Reagents, supplies, 25 chips, 2 cleaning chips, 5067-1513
low- to high- picogram range. 1 syringe, and reagent kit guide. A lower marker and
ladder are included with the RNA Pico reagents.
Small RNA Kits For analysis of small RNAs in the range of 6 – 150 nt in low- Reagents, supplies, 25 chips, 2 cleaning chips, 5067-1548
picogram sensitivity. 1 syringe, and reagent kit guide. A lower marker and
ladder is included with the small RNA reagents.
Cooled RNA 6000 Nano For use with the Agilent 2100 Bioanalyzer and RNA 6000 Nano kits. Comprises reagents and supplies box. 5067-1512
Reagents and Supplies
Frozen RNA 6000 Nano Ladder For use with the Agilent 2100 Bioanalyzer and RNA 6000 Nano kits. Comprises frozen Nano ladder. 5067-1529
Cooled RNA 6000 Pico For use with the Agilent 2100 Bioanalyzer and RNA 6000 Pico kits. Comprises reagents and supplies box. 5067-1514
Reagents and Supplies
Frozen RNA 6000 Pico Ladder For use with the Agilent 2100 Bioanalyzer and RNA 6000 Nano kits. Comprises frozen Pico ladder. 5067-1535
Cooled Small RNA Reagents For use with the Agilent 2100 Bioanalyzer and small RNA kits. Comprises reagents and supplies box. 5067-1549
and Supplies
Frozen Small RNA Ladder For use with the Agilent 2100 Bioanalyzer and small RNA kits. Comprises frozen small RNA ladder. 5067-1550

DNA Solutions Description Contents Catalog No.

DNA 1000 Kits For high resolution sizing and quantitation of dsDNA fragments Reagents, supplies, 25 chips, 1 cleaning chip, 1 syringe 5067-1504
from 25 to 1000 bp. and reagent kit guide.
DNA 7500 Kits For accurate sizing and quantitation of dsDNA fragments from 100 Reagents, supplies, 25 chips, 1 cleaning chip, 1 syringe 5067-1506
to 7500 bp. and reagent kit guide.
DNA 12000 Kits For sizing and quantitation of dsDNA fragments from 100 to 12000 bp. Reagents, supplies, 25 chips, 1 cleaning chip, 1 syringe 5067-1508
and reagent kit guide.
Cooled DNA 1000 reagents and For use with the Agilent 2100 Bioanalyzer and DNA 1000 kits. Comprises reagents and supplies box. 5067-1505
supplies
Cooled DNA 7500 reagents and For use with the Agilent 2100 Bioanalyzer and DNA 7500 kits. Comprises reagents and supplies box. 5067-1507
supplies
Cooled DNA 12000 reagents For use with the Agilent 2100 Bioanalyzer and DNA 12000 kits. Comprises reagents and supplies box. 5067-1509
and supplies
Protein Solutions Description Contents Catalog No.
Protein 230 Kits For rapid sizing and analysis of proteins from 14 to 230 kDa. Reagents, supplies, 25 chips, 1 cleaning chip,1 syringe 5067-1517
and reagent kit guide.
Protein 80 Kits For rapid sizing and analysis of proteins from 5 to 80 kDa. Reagents, supplies, 25 chips, 1 cleaning chip, 1 syringe 5067-1515
and reagent kit guide.
High-Sensitivity Protein 250 For rapid sizing and analysis of proteins from 10 to 250 kDa in Reagents, supplies, 10 chips, 1 cleaning chip, 1 syringe 5067-1575
Kits silver staining range (down to low pg/ml). and reagent kit guide.
Cooled Protein 230 and For use with the Agilent 2100 Bioanalyzer and Protein 230 kits. Comprises reagents and supplies box and appropriate cool pack. 5067-1518
Supplies
Cooled Protein 80 and Supplies For use with the Agilent 2100 Bioanalyzer and Protein 80 kits. Comprises reagents and supplies box and appropriate cool pack. 5067-1516
Cooled High-Sensitivity Protein For use with the Agilent 2100 Bioanalyzer and HSP-250 kits. Comprises all required separation and labelling reaction 5065-1576
250 and Supplies reagents.
Cooled High-Sensitivity Protein For use with the Agilent 2100 Bioanalyzer and HSP-250 kits. Comprises labelling reagents. 5065-1577
250 Labelling Reagents
Cooled High-Sensitivity Protein For use with the Agilent 2100 Bioanalyzer and HSP-250 kits. Comprises ladder only. 5065-1578
250 Ladder
Cell Solutions Description Contents Catalog No.
Cell Kits For analysis of cell fluorescence parameters. Reagents, 25 chips, and reagent kit guide. 5065-1519
Cell Checkout Kits For testing proper cell assay operation. Reagents, 7 checkout chips, red beads, blue beads 5065-1520
and reagent kit guide.
Cell Assay Extension Required to perform simple flow cytometric analysis with the Agilent 2100 Bioanalyzer (for “B” and “C” series instruments only). G2948CA
Includes pressure cartridge, cell fluorescence software, cell fluorescence, checkout kit, cell test chip kit, and start-up service.

15
 * This is a Licensed Real-Time Thermal Cycler(s) or www.stratagene.com
Licensed Real-Time Temperature Cycling Instrument(s)
under ABI’s United States Patent No. 6,814,934 and
corresponding claims in non-U.S. counterparts thereof, Ordering Information
for use in research and for all other applied fields except
human in vitro diagnostics. No right is conveyed expressly,
by implication or by estoppel under any other patent claim. U.S. and Canada:
1-800-424-5444 x3
Absolutely mRNA, AffinityScript, MxPro, and SideStep are
trademarks of Stratagene in the United States. [email protected]
Absolutely RNA, Brilliant, FullVelocity, Mx3000P, and
Mx3005P are registered trademarks of Stratagene in the
United States.
Asia Pacific:
[email protected]
Agilent is a registered trademark of Agilent Technologies.
Excel® and PowerPoint® are registered trademarks of the
Microsoft Corporation. Europe:
SYBR® is a registered trademark of Molecular Probes. [email protected]
Scorpions™ is a trademark of DxS Ltd.
TaqMan® is a registered trademark of Roche Molecular
Systems, Inc. Distributors
TRI Reagent® is a registered trademark of Molecular For a list of worldwide distributors,
Research Center, Inc. please visit our website
FAM™, TET™, HEX™, VIC™, JOE™, TAMRA™, and ROX™ are
trademarks of Applera Corporation or it’s subsidiaries www.stratagene.com
in the US and certain other countries.
Cy™3 and Cy™5 are trademarks of Amersham Biosciences.
RiboGreen® is a registered trademark of Molecular Probes. This information is subject to change without notice.

a. Patents Pending © Stratagene, an Agilent Technologies company, 2009

b. Use of labeling reagents may require licenses from Printed in USA, January 01, 2009
entities other than Stratagene. For example, use of 5990-3495EN
fluorogenic probes in 5’nuclease assays may
require licenses under U.S. Patent Nos. 6,214,979,
5,804,375, 5,210,015 and 5,487,972 owned by
Roche Molecular Systems, Inc. and under U.S.
Patent No. 5,538,848 owned by Applied Biosystems.
TaqMan® is a registered trademark of Roche Molecular
Systems, Inc

Stratagene Products