real time pcr and digital pcr - vhir and digital pcr_applied... · • taqman® mutation detection...

The world leader in serving science1 10/12/12

Bea CabotField Application SpecialistThermo Fisher Scientific

Real Time PCR and Digital PCR

2

• Real Time PCR:

• Introduction: TaqMan™ MGB probes and SYBR™ Green dye

• Applications:

• Absolute Quantification

• Relative Quantification: Gene Expression, microRNA,

Protein Quantification, CNV and Mutation Detection

• Genotyping: Allelic Discrimination

• TaqMan Array Cards

• Digital PCR - Introduction

Agenda

3

• Common: • Uses same basic components (dsDNA, primers, dNTPs, PCR

buffer, Taq polymerase, etc.).

• PCR reactions are cycled in a temperature block

• Target quantity is theoretically doubled after each cycle.

• Different:

• Some fluorescence is added to the PCR mix.

What PCR and qPCR have in common ?What is different?

4

What is different?

Plateau phase

Linear phase

Exponential phase

Cycles

PC

R P

rodu

ct

5

What is different

Variable plateau phase

96 Replicates

Cycles

High precision during exponential phaseP

CR

Pro

duct

6

Signal Generation with TaqMan Probe

•The 5′-Nuclease assay

•This method uses 2 principles:

• FRET Technology

• 5′- Nuclease activity of the Taq polymerase

7

5’ Nuclease Assay Using TaqMan Probes

ReversePrimer

Dyes: FAM™, VIC™

ForwardPrimer

TaqMan MGB Probe

3’

5’3’

5’

68 - 70°C

5’

5’

60°C

60°CRR NFQ

8

5’3’

5’5’3’

5’

R Q

• Fluorescence Resonance Energy Transfer (FRET) from high energy (Reporter) to low energy (Quencher) dye

• No reporter signal with intact probe


9

• Cleavage of probe by 5‘ nuclease activity of Taq polymerase

• FRET disabled, generation of reporter signal

5’3’

5’5’3’

5’

Q

R


10

• SYBR® Green I dye fluoresces upon binding to double-stranded PCR product

• Signal is increasing by 1000 fold when binding

• Emitted fluorescence is proportional to amount of amplified product detected in every cycle

Signal Generation with SYBR Green Dye

excitation : 497 nmemission : 520 nm

11

• Can monitor non-specific fluorescence using dissociation curve analysis after PCR (slow heating from 60° to 95°C)

• “Melting” of ds DNA reduces fluorescence

• Sharp, single peak indicates specific amplification

• Diffuse or multiple peaks indicate nonspecific amplification

SYBR Green I Dye: CHECKING FOR SPECIFICITY

Dissociation Curve

Dissociation Curve (Derivative)

Dissociation Curve (Derivative)

One, clean peak = no extraneous products

12

-

TaqMan probes

+•• More specific

• No concern about dimers

• Allows for multiplexing

• Minimal optimization

• Millions of pre-developed

Assays!

•• Can be more expensive

TaqMan or SYBR Green I Dye?

•• Can be cheaper

•• Less specific• Must run melt curves • No multiplexing / Optimization

SYBR Green I

-

+

13

Gene Expression TaqMan AssaysXS:

75/150 rx for ONLY 79.25€

1 rx- 1 € (Vol 20ul)

1 rx- 0.5 € (Vol 10ul)

14

Threshold

CT value

Rn

CyclesBaseline

Let‘s go over Terminology

Amplification Plot Terminology

15

Applications

Plus/minus

Allelic Discrimination

Relative Quantification

AbsoluteQuantification

QuantitativeApplications

End-Point Applications

16

• Forensic labs: Knowing if there is any amplifiable DNA is

necessary to decide if further investigation on sample makes

sense or not

• Microorganism Quantitation: Viruses, Bacteria, Fungi,

Parasites

Applications

AbsoluteQuantification


17

Ct‘s

Log copy number

Ct v

alue

s

0 1 2 3 4 5 6 7

The well contains 400 target copies

18

TARGET

• Gene Expression: mRNA

• Fusion transcripts: mRNA

• microRNA Analysis: MicroRNA

• Protein Quantification: Protein

• Copy Number Variation (CNV): gDNA

• TaqMan® Mutation Detection: gDNA

Applications



19


Quantification

Relative quantificationAbsolute quantification

• Results are copy numbers, ng,.. • Results are ratios between samples or biological groups named

• Relative Expression, RQ or fold change

• Numbers show increase or decrease

20

• What is a translocation and why is that important?

Fusion transcripts

� Translocations can cause Cancer

� Example: chronic myeloid leukemia (CML) & Philadelphia Chromosome

� Sometimes a fusion transcript between two genes occurs (e.g. BCR-ABL)

� Fusion transcripts are complex, and difficult to study

21

Fusion transcripts

22

• Detection from 10-500 cells or 1-1000 ng protein from tissues• Combines protein detection using Antibodies with robust, sensitive real-

time PCR• Relative quantification of proteins in cell and tissue lysates

• no purification of proteins required!• Just lyse and dilute….

TaqMan Protein Assays

• Applications:− Small sample protein analysis− Correlation of RNA & Protein

> miRNA:protein

> mRNA:protein− Validation of siRNA induced silencing− Validation of Gene Transfection/Transduction− Sample analysis from FFPE & Frozen Tumor tissues− Analysis of in vitro protein:protein interactions

23

• Assay probes are made from biotinylated-antibodies & streptavidin-linked oligonucleotides

Single tube of biotinylated polyclonal antibodies- many specificities

Combine for Binding in Assay

TargetTarget

Connector oligoSASA BBB

SASABBB

++SASA

3’

Streptavidin-oligo

SASA

5’Streptavidin-oligo

BBB B

BBB

BB

BBB B

BBBBB

BBB B

BBB

BB

Split in half

Assay probe B

SASA5’

BBB

Assay probe A

SASAB

BB

3’


24

TargetTarget

SASAB

BB

SASABBB

3’ 5’

Connector oligo

DNALigase

vs.

Connector oligo

DNALigase

SASA5’

BBBSASA

BBB

3’

TargetTarget

TargetTarget

SASA BBB

SASABBB

primer

probe

primer

• Ligation + qPCR• Protein Target-specific amplification & signal

No LigationNo qPCRNo Target-specific signal


25

Data AnalysisLigationBindingPrepare Sample

Target

3’

BBB B

BBB

BB

3’ 5’

Target

connector

BBB

0

0

1

10

100

1 4 10 14 28

Day

Fol

d C

hang

e

0.1

0.01

TaqMan® Fast Real-Time PCR

TaqMan® Protein Assay Overview

Cycle number

Lyse cells and

dilute into plate

Only 2 uL sample

required!

Bind Assay Probes

(2 uL) to protein in

crude lysate

Ligate Oligos after

binding (100 uL)

Run Fast Real-

Time PCR (20 uL)

Analyze Data for

Relative Quant

Compatible with StepOnePlus TM, 7500, 7500Fast, 7900HT, & ViiA TM 7 Real-Time PCR Systems

~3.5 hrs

~1.5 hrs

Total Time

Time to Results

Hands on Time

~3.5 hrs

~1.5 hrs

Total Time

Time to Results

Hands on Time


26

ProteinAssist Software

27

From Nature, Vol. 437 (Oct. 20, 2005)

• CNV is one of the important structural variations in the genome

• Represents a copy number change involving a DNA fragment that is ≧ 1 kb

• CNV may account for a significant proportion of normal variation and also disease association

• Genetic diseases, cancer, neurological disorders, and immune diseases

• CNVs have impacts on levels of gene expression and therefore produces gene dosage effects

Copy Number Variations

28

• PRE-DESIGNED ASSAYS (FAM-MGB)

• >1.6 million pre-designed TaqMan Copy Number Assays available

• Genome-wide coverage (~ 1 kb average spacing) including:

• known genes (~ 95% RefSeq genes covered; ~ 50% of assays)

• known CNVs (~ 85% of CNVs in DGV Nov 2008 release; ~ 25% of assays)

• Extragenic and non-known-CNV regions (~ 25% of assays)

• CUSTOM DESIGN PIPELINE AVAILABLE (FAM-MGB)

• COPY NUMBER REFERENCE ASSAYS (FAM-TAMRA)

• Human: RNase P – recommended as first choice

TERT - option in event of chromosome 14 abberation or other issue

• Mouse: Tfrc – recommended as first choice (Transferrin Receptor on Chr. 16)

Tert - alternative (Telomerase Reverse Transcriptase on Chr. 13)

Copy Number Variations - Assays

29

TaqMan CopyCaller Software

30

• TaqMan® Mutation Detection (MD) Assays using castPCR™ technology

• Higher specificity for SOMATIC mutation detection: all assays can detect

100 copies of mutant allele in the background of 104 copies of wild type

allele (1% mutation); 80% assay can detect 10 copies of mutant allele

(0.1% mutation)

• TaqMan® Mutation Detection Assays applications:

• Screening and mutation profiling of research cancer samples

• Validation of results from NGS platforms

TaqMan Mutation Detection

31

Mutant allele assay

• An allele-specific primer detects the mutant allele

• An MGB oligonucleotide blocker suppresses the wild type allele

Gene reference assay

• A locus-specific primer pair amplify a mutation-free region of the target gene

• Contain TaqMan® FAM™ dye-MGB probes

Two Main Types of TaqMan Mutation Detection Assays

ASP – allele specific primerASB – allele specific blocker (MGB oligo)LST – locus specific TaqMan ® FAM™-MGB probeLSP – locus specific primer

LST – locus specific TaqMan ® FAM™-MGB probeFP – forward primerRP – reverse primer

32

Mutation Detection Analysis Results Table

33

• Single Nucleotide Polymorphism (SNP)• Base substitution involving only one single nucleotide

• ~10 millions are thought to be present in the human genome at >1%, leading to an average of one SNP difference per 1250 bases between randomly chosen individuals (The International HapMap Consortium (2003))

Applications

Allelic Discrimination

End-Point Applications

34

Allele 1

5’-GTCGTACGTCAGTCCG-3’

3’-CAGCATGCAGTCAGGC-5’

Allele 2

5’-GTCGTACTTCAGTCCG-3’

3’-CAGCATGAAGTCAGGC-5’

• Single Nucleotide Polymorphism (SNP)

• Usually two alleles present (“bi-allelic markers“)

• MAF > 1%, if not it is called a “rare mutation”

What is a SNP?

35

Presence of Allele

1

R Q

PMGB

Q

PMGB

R

Presence of

Allele 2Q

PMGB

Q

PMGB

R

R

Allelic Discrimination Application

36

Allele calling: end point analysis

Genotype Allele Signal

FAM™ dye VIC® dye

Homozygous 1/1

Homozygous 2/2

Heterozygous 1/2

37

• TaqMan® Genotyper™ Software : Standalone and free data analysis software for Applied Biosystems® TaqMan® Genotyping Solutions

• Thermo Fisher Cloud

Allelic Discrimination Analysis

38

TaqMan Genotyper Software: Results

39

Gene Expression Studies

40

• Measurement of the concentration of the gene target in the sample

using arbitrary units

• “Relative” means the results have no meaning unless compared to

other samples

• Relative quantification allows sample to sample, but not gene to

gene quantity comparisons


41

• Difference to absolute Quantification:• No standard curve needed!

• Calculation of results by comparison of Ct values• Method used: “Comparative Ct method“

• Based on assumption that genes used in the study show similar PCR efficiencies and

• Amplification efficiency is close to 100% as in formula 2-∆ ∆ Ct is used

• Normalization with:• Endogenous Control

• Calibrator (or Reference Sample)

Relative quantification

42

• Endogenous control gene: allows for the normalization of the target's gene

expression to another gene that is constantly expressed across samples.

• Usually, the endogenous control is used to normalize for differences in the

amount of cDNA used in the PCR reaction as well as any PCR inhibition caused

by the sample.

• Choosing the appropriate endogenous control will depend upon several factors.

• Scientists have to validate their own samples to see which candidate control is stably

expressed across all of your samples.

• You can find more information on the expression of common human and mouse

control genes in the application note:

https://tools.lifetechnologies.com/content/sfs/brochures/cms_042279.pdf

What is an endogenous control, and how do I pick one?

43

Normalization with Endogenenous Control Gene

• Single Endogenous Control (TaqMan® Assays)• Select ”study-representative” samples

• Run RT with equal amounts of RNA in parallel, load equal amounts of cDNA

• Choose gene with little / no variation

• Tip: Try other candidates if no gene deliversacceptable results

• Alternatively - Run several candidates throughout the entire study and choose normaliser at the end

• Often done e.g. on TaqMan® Arrays

44

• Simultaneous evaluation of 32 candidate endogeneous controls

• Pre-loaded primers and TaqMan® probes at optimized concentrations

Testing Expression of Candidate Genes - Setup

Sample 1 Sample 2 Sample 3

45

• expression levels of reference gene and gene of interest do not need to be similar but …

• First criteria to choose an endogenous control should be always the gene expression stability across samples.

• When working in multiplex, it is recommended you choose those candidates that show similar expression to target genes, in order to not inhibit target gene amplification.

• Working in singleplex, it is recommended you avoid those endogenous controls with vey low Ct values, which require an intermediate sample dilution to increase Ct values. This step increases error in the final measure.

Misconception for normalization

46

• Evaluation of 10 commonly used endogenous control genes in 5 different tissue panels

http://medgen.ugent.be/~jvdesomp/genorm/

Algorithms of Normalization- geNorm

47

Normalization Strategies

• Endogenous Control (Reference Gene)

• Single Endogenous Control

• Multiple Endogenous Control

• Global Normalization

• Spike-In

48

• What is the Function of a certain gene ?

• What happens when I knock it down ?

Example for relative quantificaton experiment

49

Validate si-RNA directed gene knock down

Silencer® Select siRNATransfection Reagent (e.g. Lipofectamine® /

Electroporation

Cells-to-Ct™ kit• Lysis of cells directly in 96well plate• Stop lysis • Set up Reverse Transcription reaction• Real-Time PCR

50

Calibrator/ Reference Sample

timet=0 t=48h t=1 week t=2 weeks

total RNA

cDNA

total RNA

cDNA

total RNA

cDNA

total RNA

cDNA

Relative quantification (RQ) experiment to check knock down

51

∆Rn

CyclesCt =14 Ct = 24

∆Ct = 24 – 14 = 10

Endogenous controlTarget gene

Comparison of target and endogenous control

52

∆Rn

CyclesCt=15 Ct=33

t=48h∆Rn

CyclesCt=15 Ct=30

t=0

∆Rn

Cycles

Ct=9 Ct=24

t=2 weeks∆Rn

CyclesCt=14 Ct=32

t=1 week

Endogenous control Target gene

Comparative Ct method: an example using the four samples

53

Comparative Ct method calculation steps

2. Normalization to calibrator/reference

DCt Sample – DCt Calibrator = DDCt

1. Normalization to endogenous control

Ct Target gene – Ct Endogenous control = DCt

For calibrator/reference and all other samples

3. use the formula

2-DDCt

54

CyclesCt=15 Ct=33

t=48h

CyclesCt=15 Ct=30

t=0

CyclesCt=9 Ct=24

t=2 weeks

CyclesCt=14 Ct=32

t=1 week

∆∆∆∆Ct=15 ∆∆∆∆Ct=18∆∆∆∆Ct=18 ∆∆∆∆Ct=15

∆Ct = Ct [Target]- ∆Ct [Endogenous control]


Comparative Ct method

55

2 - ∆∆∆∆∆∆∆∆CT =1

Normalized expression relative to t=0 = 2 - ∆∆CT

2 - ∆∆∆∆∆∆∆∆CT =0.125 2 - ∆∆∆∆∆∆∆∆CT =0.125 2 - ∆∆∆∆∆∆∆∆CT =1


CyclesCt=15 Ct=33

t=48h

CyclesCt=15 Ct=30

t=0

CyclesCt=9 Ct=24

t=2 weeks

CyclesCt=14 Ct=32

t=1 week

∆∆ Ct=0 ∆∆ Ct=3∆∆ Ct=3 ∆∆ Ct=0

Comparative Ct method

56

Relative quantification result of the 4 samples

0

0,2

0,4

0,6

0,8

1

1,2

Samples

x-fo

ld c

hang

et=0

t=48h

t=1 week

t=2 weeks

Reference sample

t=0

87.5 %Downregulation

Calculation of knock downe.g.

Result from RQ = 0.125 fold change

100 * (1 - 0.125) =

87.5%

1 / 0.125 = 8-fold down regulation

57

• MicroRNAs (miRNAs) are short 18-25 nt RNA molecules, found in abundance in plants and animals

• miRNAs are unique & ubiquitous post-transcriptional regulators that bind to complementary sequences on target mRNAs, resulting in translational repression and gene silencing

• miRNAs are well conserved in eukaryotic organisms and are thought to be a vital component of genetic regulation

• the human genome encodes about 2000 miRNAs,

• each miRNA typically regulates multiple mRNAs, and many mRNAs are regulated by several miRNAs.

miRNA analysis

58

MicroRNA Implications

DevelopmentDevelopment

TimingLee et al. 1993

Cell proliferation Brennecke et al.

2003

NeuronHatfield et al., 2005

Cell deathCell death

Baehrecke 2003Chen et al. 2006

DiseasesDiseases

Cancers Calin et al. 2002;

2004Lu et al. 2005

Voorhoeve et al. 2006

DiabetesPoy et al. 2004

EpigeneticsEpigenetics

Bao et al. 2004Henderson et al.

2006

59

*mature active strand

• Primary miRNA transcripts processed into precursor hairpin miRNA (pre-miRNA)

• Pre-miRNA transport into cytoplasm & processed by Dicer

• Mature miRNA bound by RISC complex (RNA Induced Silencing Complex)

• Bind to complementary mRNA & suppress protein expression

• Results in translational repression or mRNA degradation, depending homology

• Regulate as much as 50% of genome & implicated in many disease states

MicroRNA Biogenesis

*

60

� TaqMan® Assays provide a highly reliable way to measure expression of various RNA transcripts.

� Provides high sensitivity, specificity, dynamic range & superior data quality

� Delivers faster time-to-results

� Requires no design expertise

� Works under universal cycling conditions

� Detects only Pri-miRNA, miRNAor non-coding RNA transcripts

Pri-miRNA, miRNA & All Non-coding RNA TaqMan® Assays

TaqMan® MicroRNA

Assays

TaqMan® Pri-miRNA

Assays

61

Analysis of Gene Expression and MicroRNA Studies:ExpressionSuite SoftwareThermo Fisher Cloud

62

What is ExpressionSuite Software?

• Free web-downloadable software package− http://www.lifetechnologies.com/us/en/home/technical-resources/software-

downloads.html

• System Requirements:− Windows® XP with Service Pack 3 or Windows® 7, SP1

• Utilizes ∆∆Ct run files (.sds or .eds) to create a study file

• Compatible with the following data collection software versions / qPCRinstruments (no AB7000)

63

Features

• ∆∆Ct method is used to calculate RQ values

• Normalization

• Enables endogenous control (EC) selection (one or more)

• Viewing EC expression across all samples / groups

• Enables Global Normalization

• Flag summary for quality control

• Option to exclude outliers automatically using modified Grubbs’ test

• Easily analyze over 100x 384-well array plates or cards or 10 OpenArray® plates in one study

64

Selection of Endogenous Control(s): Control Plot

• Displays Ct values of candidate controls for all samples as well as the calculated “stability score” (geNorm/geNormPlus).Determines the best EC

65

• Box Plot - Quickly ID outliers in biological groups

• Use Flags to filter out aberrant data

• Signal Correlation plot - Assess Ct correlation between samples or bio groups by looking at shades of color and patterns on the plot. Think overview.

• Hone in on correlation details with Scatter Plot.

Quality Check (QC) Plots

66

• Gene Expression Plot – Overview of fold changes in gene expression by target or by sample / biological group

• RQ Settings Comparison – Compare results in parallel using different analysis settings.

• Volcano Plot – Quickly assess biologically & statistically significant data points in one plot.

• Heat Map - Birds of a feather flock together. Easily view relationships between samples and gene expression patterns

Results Plots

67

Gene Expression Data

68

Results Plots: Heat Map - Clustering Analysis

• What do you do once you have a list of significantly differentially expressed genes from your experiment?• Sometimes, it’s useful to look at how various subsets or groups of genes

change in different experimental conditions

• We can do this using different types of clustering analysis

What to cluster?−Genes:

• Identify groups of genes that have correlated expression profiles

−Samples:• Put samples into groups with similar overall gene

expression profiles

• Displays similarity in expression patterns and relationships of samples and genes

69

Recommendations for gene expression studiesby qPCR

70

Gene expression using real-time PCR

Before you set up an experiment:• Decide the type of real-time chemistry to use (TaqMan® or SYBR®).

• Select a reverse transcription method.

• RT-qPCR can be performed as a one-step or two-step procedure.

• Preamplification is needed?

• Select or design assays.• Target of interest, Specificity, Efficiency, Reproducibility

• Endogenous controls

• Singleplex PCR vs. duplex PCR

• Select a quantitation method.

• Comparative CT method, Relative standard curve method or Standard curve method

Analyzing data• View the amplification plots for the entire plate.

• Set the baseline and threshold values each sample and target/detector

• Use Expression Suite or CLOUD software

71

Gene expression using real-time PCR

• https://www.lifetechnologies.com/es/en/home/technical-resources/technical-reference-library/real-time-digital-PCR-applications-support-center/gene-expression-support.html

72

How do I know how much cDNA to use ?

• We recommend using 1–100 ng of cDNA per 20 µL qPCR reaction.

• However, the exact amount to use can best be determined by running a dilution series of

your input.

• Some genes may be expressed at low levels in your samples, in which case more input may

be required. If you do not know what level of expression to expect from your sample, you

can check if there is a known expression profile in NCBI by following these steps:

• Search for your TaqMan® Gene Expression Assay using the Assay ID at lifetechnologies.com.

• Click on the “View Details” button.

• Click on the UniGene ID hyperlink.

• Click on the “EST Profile” link under the

“Gene Expression” section.

• These data can give you an idea of what level of expression to expect from your particular

samples, or what tissue source would make a good positive control.

Gene Expression Support

73

• Technical replicates: additional data point of the exact same sample, and help to monitor for handling errors.

• Biological replicates : samples that undergo the same treatment or conditions, but came from separate source materials (i.e., two separate mice, or two separate dishes of cells).

What is the difference between technical and biological replicates?

74

• https://www.lifetechnologies.com/es/en/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting.html

Gene Expression Troubleshooting

75

• To distinguish real differences from random variability

• Our natural inclination (especially with our own data) is to conclude that

differences are real and to minimize the contribution of random variability

• Statistical rigor prevents you from making this mistake

Why do we need statistics?

76

• For medium to big Gene Expression, microRNA and

Genotyping projects:

• Taqman Array Card: is a 384-well microfluidic card

designed to perform 384 simultaneous real-time PCR

(qPCR) reactions

• This format allows for 1–8 samples to be run in parallel against 12–

384 TaqMan® Gene Expression Assay targets that are pre-loaded

into each of the wells on the card.

High-throughput platforms: TaqMan Array Card

77

• Pipet optimal amount cDNA* determined previously, and Universal Master Mix in a final volume of 100 ul for each sample reservoir

TaqMan Array Card

POP-6TaqMan®2x PCR

Master Mix

Applied Biosys

78

• 1 minute @ 1200 rpm (two times) Sealing Device ready to be loaded

Analysis of results: using ExpressionSuite Software

TaqMan Array Card

79

WHAT is Digital PCR?

80

• Less susceptible to noise• More accurate• More precise • More repeatable• Easier to analyze and interpret

Relative to analog, digital is typically

…

Example: what time is it?

AnalogApproximately

8:30pm

Digital8:28:47pm

Digital vs. Analog Measurement

81

The Evolution of PCR

82

Two Methods to Quantify

?

Answer: 672 yellow, 912 red…

Answer: same # as in reference jar

Unknown “Known”

• How many beans in the jar?

83

• Digital PCR is an analytical technique for quantification of nucleic acid samples based on PCR amplification of single template molecules, without reference to a standard curve.

• Use the number of positive and negative PCR reactions to count the number of target molecules

Digital PCR Workflow

84

• Digital PCR is a statistical technique requiring tens to thousands of

reaction replicates to accurately quantify the absolute number of starting

copies of a target nucleic acid sequence without the use of a standard

• Digital PCR analysis requires that at least some reactions within the

sample replicate group have zero copies

• Amplification is detected in reactions receiving at least one molecule and

classified as positive while no amplification is detectable in reactions not

receiving target and is conversely classified as negative. Following PCR,

the number of positive and negative reactions is counted and fit to a

Poisson distribution to estimate the absolute copies of template

molecules present in the sample volume.

What is a digital PCR experiment?

85

WHEN Should I Use Digital PCR?

86

Applications for Digital PCR

Gene Expression , miRNA & Copy Number Variation

Generation of references and standards

NGS library quantification

Absolute quantification

Rare cancer target detection

Confirmation of NGS variant detection

Allele detection

Absolute quantification of pathogen (e.g. viral load)

Detection of harmful strains of pathogens

GMO detection and monitoring

87

Copy Number Discrimination

� 20% copy number difference is easier to detect usin g Digital PCR

Real-Time PCR

Cycle

Rn

dCt ~ log2(1.2) = 0.26~ 1% difference in

signal

Digital PCR

# copies / well

20% difference in signal

15 pos.

13 pos.

20% more target in Sample A than Sample B

88

WHY I Should Use Digital PCR?

89

Key Performance Attributes of Digital PCR

Sensitivity

Single molecule detection

Specificity

Exquisite discrimination

Precision

Many counting events means high accuracy

Absolute Quantification

90

QuantStudio™ 3D Digital PCR System Digital PCR Within Reach

Affordable Priced at more than 50% less than competing platforms

Simple As little as a minute to load, a minute to read

Scalable ArchitectureChip-based platform easily scales throughput & dynamic range

91

Sealed System

AmplifyLoad ReadMix

� SEALED SYSTEM

� LIMITED HANDS-ON

� MINIMAL SAMPLE LOSS

Chip-based

Droplet-basedamplify

Simple Workflow With Minimal Sample Handling

92

Just How Small is QuantStudio™ 3D Digital PCR System

93

• Intuitive Touchscreen Functionalities

QuantStudio™ 3D Touchscreen

<1 minute

• Instant results in copies/ µL and result quality displayed on the touch-screen

94

Ability to detect the KRAS G12C Mutation at frequen cyof .025% and .01% vs. the Wild Allele

Representative datasets

Detection Of Rare Somatic Mutation Down To 0.01%

9595

For Research Use Only. Not for use in diagnostic procedures.

© 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a trademark of Roche Molecular Systems, Inc., used under permission and license. Windows is a trademark of Microsoft Corporation.

real time pcr and digital pcr - vhir and digital pcr_applied... · • taqman® mutation detection...

Documents