IntroductionToRealTimeQuantitativePCR(qPCR)

SABiosciences,AQIAGENCompany
www.sabiosciences.com

Samuel Rulli, Ph.D.

Samuel.Rulli@QIAGEN.com
support@sabiosciences.com

Sample & Assay Technologies

The Seminar Topics

Workflow&applications:RNAandDNA
qPCRforgeneexpression:Whatisthechangeingene
expressionduringdifferentiation?
FactorsinfluencingtheperformanceofaqPCRassay
RNApurityandintegrity
ReverseTranscription
qPCR,reporterchemistriesandcharacteristicsofagoodqPCR
assay
AnalyzingqPCRcurves
Data&analysis

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 What does Real-Time qPCR Stands for?

RealtimeqPCRisasensitiveandreliable methodfor
detectionandquantificationofnucleicacids(DNA,&RNA
(cDNA)levels.
Itisbasedondetectionandquantificationoffluorescence
emittedfromareportermoleculeatrealtime.
ThisdetectionoccursduringtheaccumulationofthePCR
productwitheachcycleofamplification,thusallows
monitoringthePCRreactionduringearly&exponential
phase wherethefirstsignificantincreaseintheamountofPCR
productcorrelatestotheinitialamountoftargettemplate.

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 Applications for qPCR

GeneExpressionProfilingAnalysis
RNA
miRNAExpressionProfilingAnalysis

SNPGenotyping&allelicdiscrimination
SomaticMutationAnalysis

DNA CopyNumberDetection/VariationAnalysis
ChromatinIPQuantification
DNAMethylationDetection
PathogenDetection
ViralQuantification

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 Work Flow: A Brief Look

Samples
SYBR orProbe
RNA(total,mRNA,smallRNA) DNA
Reversetranscription
AssayDesign
cDNA Samplequality
control
AssayOptimization

RealTimePCRSetUp

InstrumentSetup&thermalcycling

DataOutput&Analysis
*
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 Application example:
gene expression changes during differentiation

Osteogenesis Day 16

T4
T3
T2
hMSC T1

Neurogenesis 72 hr
T1
T2
T3
T4
Differentiation protocol
Collect Total RNA at different time points
Measure 1 HKG and 1 GOI (TNF)
Repeat experiment 3x (biological replicates)

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 Applications for qPCR

GeneExpressionProfilingAnalysis
RNA
miRNAExpressionProfilingAnalysis

SNPGenotyping&allelicdiscrimination
SomaticMutationAnalysis

DNA CopyNumberDetection/VariationAnalysis
ChromatinIPQuantification
DNAMethylationDetection
PathogenDetection
ViralQuantification

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 Work Flow: A Brief Look

Samples
SYBR
RNA(total,RNA)
Reversetranscription
AssayDesign
cDNA Samplequality
control
AssayOptimization

RealTimePCRSetUp

InstrumentSetup&thermalcycling

DataOutput&Analysis

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 Factors Critical For A Successful Assay

DNAorRNAsamplepreparation Templatequality
Chooseappropriatesamplepreparationkits/reagents(inhibitorscan
compromiseRTorPCRReaction
ReversetranscriptionforconvertingRNAtocDNA
ChooseRTkits(typeofRT,whichtypeofprimers,controls?)
Assaydesign:chemistry,specificity,PCRefficiency,&throughput&cost
Choose validatedassay,orneedtovalidateourown?
RunningPCR
Choosecommercialmastermixormakeown(primer,probe,mastermix)
Dataanalysistool
Userfriendly&streamlineddataanalysismodule

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 RNA Isolation

RNAIsolation:
Qiazol?
Columnbasedmethod(RNeasy?)
Both:EfficientlysisandinhibitionofRNases;moleculargradeRNA
miRNA?UseakitspecificformiRNAandmRNA

Qiazol:
phenol/guanidine-based lysis

Instant inactivation of RNases

Instant end of biological activities

Column cleanup:
Molecular biology grade RNA

RNeasy Lipid tissue mini Kit

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 RNA Sample Quality

Spectroscopic: measure 260/280 and 230/280

OD260 is used to calculate amount of nucleic acid
260/280 ratio (typical minimum value 1.8)
260/230 ratio (typical minimum value 1.7)
Low ratio may indicate protein, QIAzol, Carbohydrates, Guanidine HCL,
Absorbance measurements do not show integrity of RNA

Denaturing RNA Agarose Gel

Used to detect integrity of RNA (usually through ribosomal bands)

QIAxcel
Automate
RNA integrity
analysis

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 qPCR Components & Steps: Overview

A.Templates B.Primers/Probes
10 1000copiesofnucleicacids
100pgto1gRNA
C.MasterMix
DNAPolymerase
ReverseTranscription Mg++
dNTP
Buffer
*Passivereferencedye
TwostepqPCR:(1)RT(2)qPCR

OnestepqPCR:onetubereaction

Denaturation Annealing Extension

Denaturation Annealing/Extension

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 qPCR Components & Steps: Overview

A.Templates B.Primers/Probes
10 1000copiesofnucleicacids
100pgto1gRNA
C.MasterMix
DNAPolymerase
ReverseTranscription Mg++
dNTP
Buffer
*Passivereferencedye
TwostepqPCR:(1)RT(2)qPCR

OnestepqPCR:onetubereaction

Denaturation Annealing Extension

Denaturation Annealing/Extension

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 Reverse Transcription

Used to make cDNA copy of RNA

Reagents:
Reverse transcriptase many different kinds
dNTPs
Buffers for RT
Primers
Random pentamers or hexamers?
Oligo-dT?
Both?

Control RNA to monitor reverse transcription kit?

Note: Make sure that RT reaction is linear

Do not try to reverse transcript too much RNA
Sensitivity of qPCR step is dependent on good RT reaction
Monitor RT reaction to ensure equal RT efficiency across all samples

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 What is in a PCR Reaction?

PCR= Polymerase Chain Reaction

Exponential Amplification of DNA in single tube

DNA Template
(ss or ds)

Polymerase
thermostable i.e. can withstand temperatures
Up to ~95C

All reagents in
dNTPs. Excess (non-limiting)

Primers (2)

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 PCR Reaction in Action

DNA Template
(ss or ds)

Polymerase

dNTPs.
1. Heat denature template (~95C)
Primers (2) 2. Anneal Primer (~60C)
3. Extend primer (~60C)
4. Repeat (~95C)

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 PCR Reaction in Action

Heat denature
DNA Template
(ss or ds)

Polymerase

dNTPs.
1. Heat denature template (~95C)
Primers (2) 2. Anneal Primer (~60C)
3. Extend primer (~60C)
4. Repeat (~95C)

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 PCR Reaction in Action

DNA Template
(ss or ds)

Polymerase

dNTPs.
1. Heat denature template (~95C)
2. Anneal Primer (~60C)
3. Extend primer (~60C)
4. Repeat (~95C)

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 PCR Reaction in Action

Polymerase

Polymerase
DNA Template
(ss or ds)

Polymerase

dNTPs.
1. Heat denature template (~95C)
2. Anneal Primer (~60C)
3. Extend primer (~60C)
4. Repeat (~95C)

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 PCR Reaction in Action

Polymerase

Polymerase
DNA Template
(ss or ds)

Polymerase

dNTPs.
1. Heat denature template (~95C)
2. Anneal Primer (~60C)
3. Extend primer (~60C)
4. Repeat (~95C)

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 PCR Reaction in Action

Polymerase

DNA Template
(ss or ds)
Polymerase

Polymerase

dNTPs.
1. Heat denature template (~95C)
2. Anneal Primer (~60C)
3. Extend primer (~60C)
4. Repeat (~95C)

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PCR Reaction in Action

Polymerase

DNA Template
(ss or ds)

dNTPs.
1. Heat denature template (~95C)
2. Anneal Primer (~60C)
3. Extend primer (~50 to ~70C)
4. Repeat (~95C)

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qPCR Reaction: Measure DNA amount at end of each cycle to
get ratio of DNA or absolute amount (if using a standard)

Polymerase

DNA Template
(ss or ds)

dNTPs.
1. Heat denature template (~95C)
2. Anneal Primer (~60C)
3. Extend primer (~50 to ~70C)
4. Measure Amount of PCR Product
5. Repeat (~95C)
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 Real-Time qPCR Fluorescence Chemistry

DNAbindingagents
Twomostcommonlyused
SYBR IDye, chemistriesinqPCRcommunity

HydrolysisProbes

DuallabeledHydrolysis(Taqman)probe

Others,suchashybridizationprobes

Molecularbeaconandscorpionprobes

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 SYBR Green I Assay: Fluorescent DNA Binding Dye

NonfluorescentSYBRI
SYBRIbindstodoublestrandDNAbutnot
singlestrandDNA.Littlefluorescenceemitted
fromSYBRIinsolution.

SYBRIuponbindingtodoublestrandDNA
emitsfluorescenceverybrightly
Simple&costsaving
FluorescentSYBRI

TheSYBRIsignalintensitiescorrelatewith
DNAamplified(ampliconamount)thusthe
initialsample inputamounts

HighSpecificityIsRequired whenusingSYBRGreen
sinceSYBRIbindsalldoublestrandDNA(nonspecificorprimerdimmer).

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 Understanding Kinetics in PCR

AmplificationPlot(Linearscale)
Endpointdatacollection
Plateau
atplateau(gelanalysis)
FluorescenceSignal

Reactionsstartvaryingdueto
reagentdepletion&decreased
PCRefficiencies(enzymeactivity,
Moreproductcompetingforprimer
annealing

107 106 105 RealtimePCRdoesearlyphasedetectionat

theexponentialstate

Preciselyproportionaltoinputamounts

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Hydrolysis Based Probe - - - Taqman Probe Assay

Thefluorescenceofthereporterdyeissuppressed
bythequencher

Primerbindingfollowedbyextension

ProbecleavagebyTaqtofreethereporterdyethus
thefluorescenceintensitycorrelateswiththeinitial
sampleinputamounts.
Taqhas5 3 exonucleaseactivity

Eachampliconneedsasequencespecificprobe(cost&time)

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 Understanding Kinetics in PCR

AmplificationPlot(Linearscale)
Endpointdatacollection
Plateau
atplateau(gelanalysis)
FluorescenceSignal

Reactionsstartvaryingdueto
reagentdepletion&decreased
PCRefficiencies(enzymeactivity,
Moreproductcompetingforprimer
annealing

107 106 105 RealtimePCRdoesearlyphasedetectionat

theexponentialstate

Preciselyproportionaltoinputamounts

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 Characteristics of a good qPCR Assay

Amplification efficiency: 100% during exponential phase

Sensitivity: Able to detect down to reasonable quantities of template in 1

reaction (10-50 copies)

Specificity: 1 assay, 1 target: (no off-target amplification or primer-dimers)

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Amplification Analysis: standard curve and single curve
analysis

Plot:
x axis dilution
Y axis Ct value

Amp efficiency = 10(-1/slope) -1 *100

Single curve analysis

PCR Miner
http://miner.ewindup.info/versio
n2

DART
www.gene-
quantification.de/DART_PCR_
version_1.0.xls
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 Sensitivity: How many copies can my assay detect?

Sensitivity is very important for low expressed genes or where there is limited sample

Method 1: Use primers to make PCR product, T/A clone, grow-up, isolate,
quantitate and use for qPCR reactions

Method 2: Use gDNA as template and use mass of gDNA to calculate copy
number and assume 1 target per genome (or actually calculate targets using
bioinformatics)

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Specificity: SYBR Green

Single peak dissociation

curves

Single gel bands of

predicted size

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Melt Curve Analysis: The General Program Steps

Rapidheatingofamplifiedsamplesto94Ctodenaturethe
DNA
Coolingthesampleto60CtoletDNAdoublestrandsanneal
Slowlyheating(byincreasingthetemperature,usually
0.2C/sec)thesamplewhileplottingthefluorescentsignal
versustemperature.
Asthetemperatureincreases,andDNAmelts,thefluorescent
signalshoulddecrease.
Therewillbeasignificantdropofthesignalwhen50%DNA
melts.

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 Melting Curve Analysis --- Normalized Reporter Plot

Plot NormalizedReporter(Fluorescence/Passivedyesignal)
NormalizedFluorescenceSignal

Samples Tm
GeneA 77.36
Rn

GeneB 78.94
50%fluorescence
drop

Tm:A Tm:B
Temperature

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 Melt Curve Analysis --- 1st Negative Derivative Plot

Plot 1st negativeDerivativeReporter

Gene B
deltaF/deltaT(thechangerate)

Gene A
Singlemeltcurveofeachamplicon
isrequiredforspecificityvalidation!

Tm: B
Tm: A

Temperature

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Experimental Setup

Gene 1 Gene 1
sample 1 sample 1
(n=3) (n=1)

Do I need technical replicates?

Technical = variation of technique
-machine dependent
-pipetting accuracy

Do I need biological replicates?

-biological variation

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 Biological replicates are better than technical replicates

Biological Replicates: 3 different experiments

Shows variability due to experiment

Technical replicates: 3 different measurements for same step

Shows variability due to pipetting, machine, enzymes, etc.

Sacrifice Technical replicates for biological replicates, always do at least 3 to

Get fold change and p value (or other statistics such as 95% confidence interval)

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 Thermal Cycling Programs

1Instrumentdefaultmeltcurveprogram Meltcurveanalysis
(SYBROnly)

Step1
StratageneMxp3005p
Activation
Step2
Data Step3
collection Meltcurve
analysis

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Run qPCR - - - Results

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 How To Define/Set Up The Baseline

LinearAmplificationPlot
AutomatedBaselineOption
ifaninstrumenthasaadaptivebaseline
function

Definemanually:
(1)Uselinearview oftheplot
(2)Setupthebaselinereadingfrom
cycle#2tothecyclethat2cyclesbefore
Ct theearliestvisibleamplification
Baseline
(3)Usuallyabaselinefallsin315 cycles

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 How To Define Threshold

LogViewAmplificationPlot
Uselogview ofamplificationplot
Thresholdshouldbehigherthan
baseline(higherthanthenoiselevel)
ThresholdshouldatLOWER 1/3or1/2
ofthelinearphaseofamplification
Linearphase=exponentialphase
Differentrunsacrosssamplesforthe
sameexperimentsshouldhavethe
samethreshold forcomparison

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 Reference Genes (Housekeeping Genes)
For Normalization

Anychanges?
GeneofinterestAinuntreatedcells GOIAindrugtreatedcells

ReferenceGeneBinuntreatedcells RefGeneBindrugtreatedcells

Theexpressionlevelofareferencegeneremainconsistentunder
experimentalconditionsordifferenttissues

AReferenceGene isaimedtonormalizepossiblevariationsduring:
Sampleprep&handling(e.gusethesamenumberofcellsfromastart)
RNAisolation(RNAqualityandquantity)
Reversetranscriptionefficiencyacrosssamples/experiments
PCRreactionsetup
PCRreactionamplificationefficiencies

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Commonly Used Housekeeping Genes

HKGsinRT2 ProfilerPCRArrays
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 Data Analysis website

1.) Average Ct values for all gene replicates

2.) Calculate Delta Ct value between GOI and HKG for each experiment

3.) Average Delta Ct values between experiments (replicates)

4.) Calculate Delta-Delta Ct values ( Delta Ct experiment- Delta Ct control)

5.) Calculate Fold Change 2(-Delta Delta Ct)

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 Normalized Gene Expression Level

Anychanges?
TargetGeneAincontrolcells TargetGeneAindrugtreatedcells

ReferenceGeneBincontrolcells RefGeneBindrugtreatedcells

Ct=Ct(TargetAtreated) Ct(RefBtreated)

Ct=Ct(TargetAcontrol) Ct(RefBcontrol)

Ct= Ct(treated) Ct(control)

Normalizedtargetgeneexpressionlevel=2(Ct)

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 Delta Delta Ct Method: A Look of Amplification Plots

Ref GAPDH
GOI
TNF

Ct Ct Ct Ct

Ct =Ct (TNFtreatGAPDHtreat) ct (TNFcontrolGAPDHcontrol)

The fold change = 2(-Ct)
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 Data Analysis website

1.) Average Ct values for all gene replicates

2.) Calculate Delta Ct value: GOI-HKG

3.) Average Delta Ct values between experiments (replicates)

4.) Calculate Delta-Delta Ct values (Delta Ct experiment- Delta Ct control)

5.) Calculate Fold Change 2(-Delta Delta Ct)

TNF is up-regulated 32 fold in the treated cells versus the control

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http://www.sabiosciences.com/dataanalysis.php

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http://www.sabiosciences.com/dataanalysis.php

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 Questions?

AsknoworcontactTechnicalSupportM F,9AM 6PMEST

Telephone:(888)5033187
Email:support@SABiosciences.com

Thankyou!

Sample & Assay Technologies