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The LightCycler® 480 Real-Time PCR System

Unleash the Power of Real-Time PCR

Steven van Konynenburg

Field Applications Specialist
LightCycler® 480 System
Overview

1. General Description

2. LightCycler® 480 Instrument and Specifications

3. Assay Formats, Dyes, and Applications

4. LightCycler® 480 Performance

5. LightCycler® 480 Software

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The Roche LightCycler® Story
17 Years of qPCR Innovation

2012
2011
2005
2004
1998

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LightCycler® Systems
Technological (R)Evolutions

• Accuracy
• Speed
• Versatility
• Sensitivity
• Throughput
• Automation
Carousel-Based LightCycler® 480 System
LightCycler® System

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LightCycler® 480 System
Overview

1. General Description

2. LightCycler® 480 Instrument and Specifications

3. Assay Formats, Dyes, and Applications

4. LightCycler® 480 Performance

5. LightCycler® 480 Software

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LightCycler® 480 Instrument
General Architecture

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LightCycler® 480 System
96 < > 384 Switch

• Interchangeable thermal block

cycler
• Do-it-yourself, fast
• Loading help for convenience
• No service engineer required
• No re-calibration necessary
• Instrument automatically detects
and identifies block

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LightCycler® 480 Thermal Block Cycler
Speed and Accuracy

Therma-Base for
optimized heat equalization

• Homogenous temperature
distribution over the plate

• Fast PCR runs

96 wells in < 1 hour
384 wells in < 40 min

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LightCycler® 480 Instrument Heat Sink
Therma-Base

multiwell plate mount (silver)

circuit board (Peltier elements)

Therma-Base layer

cooling element

• Thin sealed vacuum vessel with working fluid in a wick structure

• Rapidly transfers heat by evaporation and condensation
Enables both rapid and accurate cycling!
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Thermal Uniformity
Instrument Comparison

LightCycler® 480 Instrument Standard Instrument

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LightCycler® 480 Optical System
Sensitivity and Homogeneity

• LED
Filter wheels
– high intensity
– broad dynamic range
– lifetime: 10 000 hrs
• CCD camera
• Five excitation filters
• Six detection filters

CCD Camera • Optimized arrangement of optical

Field lens components
Multiwell plate
• Homogeneous excitation and
fluorescence detection

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LightCycler® 480 Optical System
Path of Rays

• Homogeneous illumination and imaging due to long object-image distance.

• Pinhole to mask lateral portions of emitted light and focus on central, perpendicular portions.
• Large field lense to efficiently collect rays also from lateral wells.

Illumination
Beleuchtung Detektion
Detection

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LightCycler® 480 Optical Unit
Characteristics
5 excitation – and 6 emission filters for
Instrument II
• Easy duplexing
Bandpass (Band Width)
– Easy and unbiased performance of dual
color Hydrolysis Probes assays using Excitation • 440 nm (35)
universal color compensation objects for • 465 nm (25)
FAM/HEX (VIC) or FAM/Yellow 555 • 498 nm (40)
combinations • 533 nm (25)
• 618 nm (35)
Detection • 488 nm (20)
• Unmatched multiplexing
• 510 nm (20)
– 4 color Hydrolysis probes • 580 nm (20)
– 4 color HybProbe probes • 610 nm (20)
• 640 nm (20)
• 660 nm (95) long pass
• The long-pass filter grants access to dyes of
higher wavelength

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LightCycler® 480 System
Overview

1. General Description

2. LightCycler® 480 Instrument and Specifications

3. Assay Formats, Dyes, and Applications

4. LightCycler® 480 Performance

5. LightCycler® 480 Software

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LightCycler® Assay Formats
... Most Commonly Used

Sequence-independent formats like SYBR Green I

Sequence specific detection formats: HybProbe Probes

Hydrolysis Probes or Universal ProbeLibrary Probes

Channels, Dyes, and Detection Formats

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LightCycler® 480 System
Allows Use of All Most Currently Used Detection Formats
Detection format Excitation (nm) Detection (nm) Dye Applications

SYBR Green l 465 510 SYBR Green I Qualitative and

quantitative PCR,
Product verification

HybProbe probes 498 610 LightCycler® RED 610 Quantification,

640 LightCycler® RED 640 SNP/mutation analysis
660 Cy5 via melting curves

Hydrolysis probes 440 488 LightCycler® CYAN 500 Quantification,

465 510 FAM Endpoint genotyping
533 580 VIC, HEX, Joe, Yellow555
533 610 LightCycler® RED 610
618 660 Cy5, Cy 5.5

SimpleProbe probes 465 510 Fluorescein SNP analysis

High Resolution 465 510 LightCycler® 480 ResoLight Gene/mutation

Melting Dye scanning, SNP analysis
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Sequence Independent Assay Format
SYBR Green l

Denaturation Annealing End of Elongation

• When SYBR Green I dye intercalates into dsDNA, its fluorescence greatly increases
• All SYBR Green I assays have to be verified by a Melting Curve Analysis for side
products other than the specific amplicon
• Dyes like the Roche Dye ResoLight completely saturate the double stranded nucleic
acid and can be used to detect unknown variations in a process called “high resolution
melting”

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Detection of Sequence-Specific Probe Binding
Fluorescence Resonance Energy Transfer (FRET)

Probe

FRET reporting

Hydrolysis Probes

FRET quenching

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Probe Based Assay Format
Format

Denaturation Annealing Elongation

• The unique LightCycler® HybProbe probes are 2 sequence specific, non-extendable

oligonucleotide probes labeled with different dyes close emitting light when coming in
close proximity
• As both probes stay intact after amplification, they may be used for a subsequent melting
curve analysis (e.g., SNP detection)
• The LightCycler® 480 optical system allows the usage of the HybProbe Format and allow
quadruplex multiplexing of HybProbe Probes

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Probe Based Assay Format (II)
Hydrolysis Probe Format

Denaturation Annealing Elongation End of Elongation

• Hydrolysis probes emit fluorescence when the 5´ 3´ exonuclease activity of Taq

polymerase hydrolyzes them
• Universal ProbeLibrary Probes are similar to Hydrolysis Probes
• The LightCycler® 480 optical system allows the usage of the Hydrolysis Probe Format and
allow quadruplex multiplexing. For unbiased fluorescence detection of duplex assays using
dye combinations like FAM/(VIC) HEX or FAM/Yellow 555 are supported by universal
Color Compensation Objects.

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LightCycler® 480 Multiwell Plates
Disposables

96-well plate for 10–100 µl 384-well plate for 5–20 µl Sealing foil

• Barcode labeled multiwell plates

– LightCycler® 480 Multiwell Plates clear, (96 and 384 well)
– LightCycler® 480 Multiwell Plates white, (96 and 384 well)
• Best fit for optimized heat transfer
— fast cycling times (384-wells in < 40 min, 96-wells in < 1h)
— homogenous temperature distribution in each well
• Sealing foil to prevent of evaporation and contamination
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LightCycler® 480 Reagents
Optimized for Each Application

• LightCycler® 480 SYBR Green I Master

• LightCycler® 480 Probes Master

• LightCycler® 480 Genotyping Master

• LightCycler® 480 High Resolution Melting Master

• LightCycler® 480 ResoLight Dye

• LightCycler® 480 RNA Master Hydrolysis Probes

• RealTime ready RNA Virus Master

• LightCycler® Multiplex RNA Virus Master

• LightCycler® 480 Control Kit

• LightCycler® 480 QC Kit

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Universal ProbeLibrary (UPL)
Overview

• Universal ProbeLibrary (UPL) Assays

– for gene expression quantification of a large number of organisms
– UPL comprises 165 short hydrolysis probes
• 8-9 mers, FAM-labeled
• Locked Nucleic Acids (LNAs) incorporated
– The UPL probe and the PCR primers together constitute the PCR assay for a given
target gene.
• Design UPL assays using ProbeFinder Software available free at the web-based Assay
Design Center at www.universalprobelibrary.com
• UPL assays are compatible with all real-time PCR instruments capable of detecting
fluorescein, FITC, FAM and/or SYBR Green I. Use a standard real-time PCR protocol for
hydrolysis probes.

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LightCycler® 480 System
Application Overview

• It allows measurement during amplification for quantitative or qualitative results

– Qualitative detection (Yes/No answer)
– Absolute Quantification (result: e.g., copies/ml)
– Relative Quantification (result: e.g., relative ratio)

• It allows measurements at the endpoint of PCR

– Endpoint Genotyping (allelic discrimination)

• It allows melting curve analysis subsequent to PCR amplification for

– Product identification with SYBR Green I
– Detection of known mutations using e.g., HybProbes
– Detection of unknown mutations with Gene scanning

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Amplification Analysis

Any probe type

Simple Probes

Hybridization probes dsDNA binding dye

Hydrolysis probes SYBR Green

Scorpion Probes

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Melting Curve Analysis

SYBR Green I High Resolution Melting Dye Hybridization Probes

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Multi Color Assay, Hydrolysis Probes
Color Compensation

• LightCycler® CYAN 500

• FAM
• HEX
• LightCycler® RED 610
• LightCycler® RED 670

• One target, dye per well

• All wells analyzed in all
channels
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LightCycler® 480 System
Overview

1. General Description

2. LightCycler® 480 Instrument and Specifications

3. Assay Formats, Dyes, and Applications

4. LightCycler® 480 Performance

5. LightCycler® 480 Software

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LightCycler® 480 System
Dynamic Range

• Serial dilution of plasmid DNA (1

copy - 109 copies)
• 9 replicates each
• Detection format: UPL (FAM)
• Target: GAPDH

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LightCycler® 480 Control Kit
Detection of a two-fold Difference

• 1000 vs. 2000 copies

• 24 replicates each
• detection format: UPL (FAM)

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LightCycler® 480
Detection of a two-fold Differencec

• 1000 vs. 2000 copies

• 16 replicates each
• detection format: one step, Hydrolysis Probes

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LightCycler® 480
Walk Around the Block

•detection format: Hydrolysis Probes

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 LightCycler® 480
Reproducibility

• detection format: SYBR Green I

• 44 replicates of sample

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 LightCycler® 480
Reproducibility

• detection format: SYBR Green I

• 44 replicates of sample

Average Tm = 88.798 STD=0.16

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LightCycler® 480 System
Overview

1. General Description and Specifications

2. LightCycler® 480 Instrument

3. Assay Formats, Dyes, and Applications

4. LightCycler® 480 Performance

5. LightCycler® 480 Software

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Crossing Point Determination

At what point does the amplification reaction transition from lag phase to log-linear phase?

1B 1B

1M 1M
log-phase
Lag Phase

1,000 1,000

12 3 4 5 6 7 8 10 11 12 13 14 15 16 17 18 19 20 12 3 4 5 6 7 8 10 11 12 13 14 15 16 17 18 19 20
2 2
PCR Cycle PCR Cycle

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Crossing Point Determination
The Fit Points Method

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Crossing Point Determination
The Second Derivative Maximum

90 12

Crossing Point (Cp)

80 10

70 8
Fluorescence (y)

Amplification Data (x, y)

Derivative of Fluorescence
60 6

50 4
1st Derivative dy/dx
40 2

30 0
2nd Derivative d2y/dx2
20 -2

10 -4

0 -6

-10 -8

Cycle Number (x)

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LightCycler® 480 System
Software Innovations(2)
•PCR efficiency consideration

•Calibrator-normalized PCR

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LightCycler® 480 System
Software Innovations(3)

• Standard curve calculation truly based on data

linear fit non-linear fit

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LightCycler® 480 System
Software Innovations
Without efficiency Efficiency correction Efficiency correction

correction with linear fit with non-linear fit

Adrenal Gland RNA

40 ng 1.03 1.18 1.41

8 ng 2.21 1.79 1.01

1.6 ng 6.00 4.17 1.17

Mean value 3.08 2.38 1.21

Standard deviation 2.5967 1.5799 0.2173

Coefficient of variation 84.3% 66.4% 18.0%

Test System: Total RNA from - human adrenal gland tissue (sample)
- HeLa cell line (calibrator)
Duplicate measurements for each data point
Data Analysis: Calibrator-normalized target/housekeeping ratios

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LightCycler® 480 Software
Flexible Analysis Options
• Melting Curve Genotyping :SNP • Absolute Quantification
analysis with HybProbe probes – Dynamic range: 1–1010 copies
– Robust melting curve analysis – External standard curves
– Automated annotation
– Ideal for multiplex assays and
haplotyping
• Relative Quantification
• Endpoint Genotyping – Basic analysis: one click to
– Using hydrolysis probes result
– For known SNPs in known – Advanced method for utmost
regions accuracy
– Accurate results using
efficiency correction
• Gene Scanning-HRM – Use of reference genes
AT AA
– Discovery of unknown genetic
TT and epigenetic changes
– Homo-/heterozygote
differentiation

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LightCycler® 480 Software
Overview

• New Experiment

• Macros are used to

run an experiment
automatically

• Templates

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LightCycler® 480 Software
Data Management
• Data stored in a traceable or
a research database

• Navigator for data base

entries

• Query possibility to find

information

• Export and Import functions

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LightCycler® 480 Software
User Management

• Different user levels (admin and

expert user)

• Users may be organized

in groups

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LightCycler® 480 Software
Instrument

• Connected instrument

• Block type,

• Lamp status,

• Operation log

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LightCycler® 480 Software
Detection Format

• 5 Excitation filters

• 6 Detection filters

• Roche-defined

detection formats

• User-defined

detection formats

possible

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LightCycler® 480 Software
Experiments

• Flexible run programming

• Online data display of

fluorescence and
temperature

• Create or apply Templates

All parts and settings of an
experiment (run, sample
management, analysis and
report) can be stored in
templates and easily applied to
experiments

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LightCycler® 480 Software
Sample Management (1): Subset Editor
• Subset Editor
• Sample Editor
• Plate view

• Create or apply
Template

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LightCycler® 480 Software
Sample Management (2): Sample Editor

• Subset Editor
• Sample Editor
• Plate View

• Create or apply
Template

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LightCycler® 480 Software
Sample Management (3): Plate View

• Subset Editor
• Sample Editor
• Plate view

• Create or apply
Template

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LightCycler® 480 Software
Analysis Modules
Analysis Modules

Optional Modules
– LIMS/Barcode module
– Gene Scanning

– Multiple Plate Analysis Software

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LightCycler® 480 Software
Analysis Module: Absolute Quantification

n Automated calculation method of

Crossing Points (Cp)

n Standard curve:
- Standards in the same
experiment
- import of a previous
saved standard curve object

n Create or apply Template

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LightCycler® 480 Software
Analysis Module: Tm Calling
n Automated calling of melting
temperatures (Tm)

n Improved algorithms for

- HybProbe Format
- SYBR Green I Format
- SimpleProbe Format

n Optional
- Manual Tm Editing
- Display settings

n Create or apply Template

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LightCycler® 480 Software
Analysis Module: Color Compensation

n Raw Data are generated with a

Color Compensation run

n Analyzed data are saved

as CC Object

n Color Compensation objects are

applied to dual or multi color
experiments

n Create or apply Template

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LightCycler® 480 Software
Analysis Module: Relative Quantification
-Basic Relative Quantification Analysis: automated, results with one click
-Advanced Relative Quantification Analysis: according to your needs

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LightCycler® 480 Software
Analysis Module: Melting Curve Genotyping
Detection of known variants

Genotyping with HybProbe-/Simple Probe

probes

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LightCycler® 480 Software
Analysis Module: Endpoint Genotyping
Detection of known variants

Genotyping with Hydrolysis probes

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LightCycler® 480 Software
Analysis Module: Gene Scanning
Detection of any variants

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LightCycler® 480 Software
Report
n Flexible selection of
report elements

n Different report settings

possible

n Create or apply template

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LightCycler® 480 Real-Time PCR System
Summary (1)

• Unique thermal block cycler technology for exceptional well-to-well data

homogeneity.
• Easily interchangeable 96- and 384-well thermal block cycler units.
• Optional clear or white plates, depending on your workflow and sensitivity needs.
• Advanced high-performance optical system for accurate data capturing.
• Highest flexibility with fluorescence dyes and detection formats.
• Intuitive, user-friendly LightCycler® 480 software interface.
• Versatile genotyping based on endpoint analysis or melting curves.
• Multi-function database with research and traceable modes.

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LightCycler® 480 Real-Time PCR System
Summary (2)

• Step-saving short cuts for high-throughput sample and data handling.

• Fast and easy assay setup with the new sample editor.
• Options for basic and advanced gene expression and genotyping methods.
• One-click experiment setup with options to refine results later.
• Excellent PCR sensitivity with high-value LightCycler® 480 reagents and disposables.
• State-of-the-art LIMS connectibility.
• 21 CFR part 11 compliance data protection.
• Premium customer support and instrument service

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 LightCycler® 480 System Support and Service
• Roche Diagnostics head office based in Laval, Quebec.
• Ontario Service Team (Instruments and Applications) for Life Sciences
Research and Molecular Diagnostics:

Roche Care Centre Charles Marseille

Technical Support Specialists Genevieve Major
[email protected]
1-800-870-7050
Sandra Pittet

Local Application Support Steven van Konynenburg

Field Application Specialists Mena Farag
Sandra Jeftic

Local Instrument Support Kenneth Brace

Field Support Representatives Steve Ruffell
Doing now what patients need next