qpcr applications using stratagene’s mx real-time pcr … · avierstr/principles/pcr.html pcr...

QPCR Applications using StratageneQPCR Applications using Stratagene’’s s Mx RealMx Real--Time PCR PlatformTime PCR Platform

Dan Schoeffner, Ph.DField Applications [email protected]. Services 800-894-1304

Polymerase Polymerase Chain Chain

ReactionReactionMeltAnneal primers

MeltAnneal

+

Extension/Measure

Gene of interest (Amplicon)

DNADNA

Forward and Reverse Primers

http://allserv.rug.ac.be/~avierstr/principles/pcr.html

PCR Molecular MechanismPCR Molecular MechanismExponential amplification of the original DNA sequence (template)

to create copies of part of the sequence (amplicon)

Xn=X0 (1+E)n

X = DNA concentrationX0= Starting DNA concentrationXn= DNA concentration at cycle n

E = Efficiency of PCR reaction, 0-1

2n

QQ

Influence of Reaction EfficiencyInfluence of Reaction Efficiency

Cycle #

Fluo

resc

ence

(R)

Baseline Raw Signal (R)

CCtt = Fractional PCR cycle number at which the fluorescence

intensity crosses the established threshold line.

• A 1CCtt difference between samples represents 2x2x more transcript

Ct

Threshold

Typical PCR Amplification PlotTypical PCR Amplification Plot

Ampli

ficati

on

Why is QPCR superior to PCRWhy is QPCR superior to PCR

96 technical replicates

Variability using endpoint PCR

Variability using QPCR

GelGel--based quantificationbased quantification

12

21

RealReal--time quantificationtime quantification

12

UnpredicableUnpredicable Amplification Plots with Amplification Plots with Endpoint AnalysisEndpoint Analysis

Chemistries used in QPCRChemistries used in QPCR

Alx350Alx350Alx350 FAMFAMFAM

TETTETTET HEXHEXHEX

ROXROXROX Cy5Cy5Cy5Cy3Cy3Cy3

TAMTAMTAM TxRdTxRdTxRd

JOEJOEJOE

350nm 700nm

Absorption

Lightλ

Emission

Lightλ

Fluorescence DetectionFluorescence Detection

Quantitative PCR ChemistriesQuantitative PCR Chemistries

SYBR Green

TaqMan®Molecular BeaconsLux® primersHybridization probesScorpionsTMAmplifluor® probes FRET

dsDNA Binding

Probe Based Detection

ChemistriesChemistries

SYBR green dsDNA binding dyes Pro:

■ Ease of use■ Inexpensive■ Good for high throughput screenings

lots of genes: this is your chemistry■ Great for first screens and optimization■ Can detect amplicon heterogenity

Con:■ Sequence unspecific – detects any

double strand in your reaction■ Can not multiplex reactions

1000x increase in fluorescence

Primer SelectionPrimer Selection• Try to achieve similar Tm for all primers: Ideal ~60°C.

(Future multiplexing or use of Taqman™ assays in mind)

• Forward and reverse primer should have ∆Tm <2°C

• 40-60% GC content to prevent G/C region self-hybridization

• ∆G of primer dimer/cross primer dimer formation > -4 kcal/mol to avoid stable primer dimers

• Design via software (Always use the same one):

• Always perform a BLAST search with your amplicon and primers( Specificity of the PCR)

SYBR greenSYBR green

SYBR Green ISYBR Green I™™ Thermal ProfileThermal Profile

Activation Amplification Dissociation

Raw Fluorescence [R]

Negative First Derivative [-R’(T)]

ChemistriesChemistries

R QTaq

RQ

QTaq

R

Taqman probesPro:■ Sequence specific■ Possibility to do multiplex

- have GOI and normalizer in the samewell, doing comparative quantification

Con:■ More difficult to design■ Expensive

Linear Linear TaqmanTaqman Probe DesignProbe Design

• Probe Tm 5-10°C higher than primers• ≤ 30 bp in length• No G next to reporter fluorophore• < 4 contiguous Gs• PCR blocker at 3’ end• Compatible reporters and quenchers

Linear Linear TaqmanTaqman Probe ModificationsProbe Modifications

Increase thermal duplex stability

Improve specificity

Raise Tm by up to 8°C per LNA

Allow shorter probe design (~13bp)

(www.proligo.com)

2'-O, 4'-C methylene bridge locks conformation

O

BaseO

O

PO O-

O

BaseO

O O

PO O-

DNA LNA

Introduction to Introduction to MxProMxPro softwaresoftware

Critical Setting

•Threshold •Baseline

Analysis term settingsAnalysis term settingsAlgorithmsAlgorithms

• Developed using real Q-PCR training data, establish settings and ranges

• Performs optimally for the majority of the fluorescence signal analyzed

• Allows a user to analyze the raw data using the same method over time, identify trends

• Easier to justify settings for validation, QA/QC purposes

Threshold ValueThreshold ValueSeparates the data from the noise.Separates the data from the noise.Valid for Ct calculations if placed Valid for Ct calculations if placed during exponential amplification.during exponential amplification.

3 Options:3 Options:–– Amplification based Amplification based

threshold (Minimizes threshold (Minimizes variability between variability between replicates)replicates)

–– 10 times the noise during 10 times the noise during early cycles.early cycles.

–– Manual (click and drag)Manual (click and drag)•• On exponential phase.On exponential phase.•• Lines are parallel.Lines are parallel.•• Minimize variabilityMinimize variability

ThresholdThreshold-- Amplification BasedAmplification Based

Baseline SubtractionBaseline Subtraction

-5000

0

5000

10000

15000

20000

25000

30000

35000

0 10 20 30 40

Cycle #

R

R

dR

dR1

R1

Xn=X0 (1+E)n

Assay Development and Assay Development and ValidationValidation

• Consistency, Consistency, Consistency• Initial optimization efforts should identify good

control or standard materials that you can rely upon throughout data generation

• Generate a range of acceptable Q-PCR performance data

• Controls should dictate what data is good or bad

QQ--PCR Assay Design ConsiderationsPCR Assay Design Considerations

QQ--PCR Assay ProcessPCR Assay Process

DesignDesign

SynthesisSynthesis

Test oligosTest oligos

PrimersPrimers

ProbeProbe

Enzyme,Enzyme,dNTP, MgdNTP, Mg++++

OptimizationOptimization

ValidationValidation

Run and AnalyzeRun and Analyze

Experimental DesignExperimental Design

Oligo DesignOligo Design

Gel

Experimental DesignExperimental DesignReplicates

nBiological Depends on biological

variability

(CV/Power Analysis)

Technical(qPCR)Reflects experimental error

(n=3 is sufficient)

Independent experiments

Ensures biological relevance

(n=2 is sufficient)

Concordance of Results?

General Strategy for New QPCR General Strategy for New QPCR Assay DevelopmentAssay Development

• Plan to optimize assay using SYBR Green chemistry

– SYBR melt curve will yield PCR specificity info that probe based detection will not

– Attempt to constrain assays to a common thermal profile for convenience

• Design amplicons compatible with probe chemistry for possible future use in a multiplex QPCR format

Components of a Quality QPCR AssayComponents of a Quality QPCR Assay

• QPCR amplification (Ct Linearity & Reproducibility, Efficiency, Sensitivity)– Standard curve should be run during assay

optimization– High efficiency correlates with sensitivity of

detection– Establishes a measuring range of assay

• PCR amplification specificity from dissociation curve (Tm)

First Assay First Assay -- Testing Testing OligosOligos

• First assay should be a standard curve run to test primers and overall assay performance– Dilution series, (1:5) X 6 points in triplicate, negative

controls– 150 to 300nM primers, ~100 ngs of template

(25nM to 1000nM) (25ngs to 250ngs)

Assay ValidationAssay ValidationStandard Curve Metrics

Quantity

Ct

Serial dilution of a positive control or amplified target

LogQuantity

Ct

Standard Curve

%Eff =10[-1/slope] -1

Expect:High efficiency (E = 90 – 110%)Good linear fit (R2 > 0.98)3-5 logs of dynamic range~1 % CV variation among triplicates

Log transform


Eff=105.6%R2=0.97


Eff=105.0%R2=1.011.5-30.2 Cts

Copies

Select the best performing and most appropriate range for samples to be run

Primer SelectionPrimer Selection• Try to achieve similar Tm for all primers: Ideal ~60°C.

(Future multiplexing or use of Taqman™ assays in mind)

• Forward and reverse primer should have ∆Tm <2°C(SYBR: 75 – 400, 200bp ; Taqman 75-150, 125bp)

• 40-60% GC content to prevent G/C region self-hybridization

• ∆G of primer dimer/cross primer dimer formation > -4 kcal/mol to avoid stable primer dimers

• Design via software (Always use the same one):

• Always perform a BLAST search with your amplicon and primers( Specificity of the PCR)

Assay OptimizationAssay OptimizationPrimersPrimers

Tm of primers depends on concentration:perform a primer matrix test to identify optimal

concentration using SYBR chemistry

50 nM 100 nM 150 nM 300 nM 600 nM

50 nM

100 nM

150 nM

300 nM

600 nM

SYBR based

What if I can not identify primers What if I can not identify primers sequences in my gene of interest sequences in my gene of interest

that have the same annealing temp?that have the same annealing temp?

Assay OptimizationAssay OptimizationPrimersPrimers

positivecontrols∆Ct = 3 NTCs

Primer titration 50 nM – 200 nMduplicates for pos. Control & NTC

Aims:low Ct values

sensitivity

no unspecificamplification orprimer dimers

specificity

Low interreplicatevariability

high efficiency ofamplification

100/150NTC

100/150

150/100NTC

150/100

QPCR Assay ControlsQPCR Assay Controls

Initial efforts should identify good control materials to run during assay setup and validation– Establish a range of acceptable QPCR

performance data– Controls will dictate what data is good or

bad and what should be included in down-stream analysis.

– Justification for omitting data or re-assay

QPCR ASSAY CONTROLSQPCR ASSAY CONTROLS

Review the most common controls to include in any QPCR experiment– Systematic Experimental Error Control– Positive QPCR controls – Negative QPCR controls

QPCR Assay ControlsQPCR Assay ControlsPassive Reference FluorPassive Reference Fluor

Passive Reference Fluor (ROX) spiked into QPCR master mix at outset of assay setup– Rox fluor emission used to correct for

artifacts in signal measurement from wells• Bubbles in sample volumes, plasticware

inconsistency, variation in sample volume.– Include Rox, measure signal, assign it as

the Reference Dye in Mx software setup– Will improve data uniformity and reduce

correlation of variance (%CV) among technical replicates

QPCR Assay ControlsQPCR Assay ControlsPassive Reference FluorPassive Reference Fluor-- ExampleExample

Signal uniformity across 96 replicate wells


~8% CV of Raw (R) Rox Signal across 96 wells


~8% CV of Raw (R) Fam Signal across 96 wells


<1% CV of Ct for Roxnormalized, baselinesubtracted (dRn) across 96 wells

QPCR Assay ControlsQPCR Assay ControlsPositive QPCR ControlPositive QPCR Control

Positive Controls- Common Sources of material– Pooled RNA/cDNA unknowns from experiments– Linearized/nicked plasmid cDNA– Purified PCR product– Stratagene Reference RNAs

QPCR Assay ControlsQPCR Assay ControlsPositive QPCR ControlPositive QPCR Control

Positive Controls- Some sample that contains your gene of interest (GOI) and should be detected by QPCR– Ideal control should be similar to the unknowns

you will be analyzing, ie RNA in same matrix as tissue or cell samples

• No Template controls (NTC)– No cDNA added to QPCR reaction– Detects primer dimer, contaminating template,

or probe degradation across cycles• No Reverse Transcription Control (NoRT)

– RNA sample undergoing reaction w/o RT– Detects contaminating gDNA in RNA

• No Amplification Control (NAC) – No Taq DNA polymerase added to QPCR

reaction– May indicate high background

QPCR Assay ControlsQPCR Assay ControlsNegative QPCR ControlsNegative QPCR Controls

QPCR Assay Control SpecificityQPCR Assay Control SpecificityNegative QPCR ControlNegative QPCR Control

Standard

Deriviation of fluorescence (R’T)Melting Temp. (Tm)


Sample

NTC, NoRT

Standard


Primer dimers

NTC

BAD !

QPCR Assay Control Specificity QPCR Assay Control Specificity Negative QPCR ControlNegative QPCR Control

Standard



NTC

NoRT

BAD !

gDNAPrimerdimers

• Assay controls are the main determinant of data quality

• Provide leverage for troubleshooting, allows you to regain assay performance quickly

• Easy to prepare, requires up-front effort, worth the work in the long term

QPCR Assay ControlQPCR Assay ControlSummarySummary

QPCR Listserver– [email protected]

Contact Stratagene Technical Services– (800) 894-1304, Pacific Standard Time– [email protected]

Webinars and Introduction to QPCR Guide:– www.stratagene.com/fasttrack– An Introduction to Stratagene's Mx QPCR Software – Principle of Quantification by Real-Time PCR – Assay Validation and Optimization – Basic Assay Troubleshooting – QPCR Assay Controls – Critical Components of Assay Design – Enhancements offered in Stratagene’s MxPro™ QPCR Software

Normalizer

Control Unknown

Gene of Interest CtCt

CtCt

∆CtGOI

∆CtNorm

Norm. ratio

(1+E) [CtC-Ct

U]

Comparative QuantificationComparative QuantificationGiven two samples: What is the difference in gene expression?

(1+E) [CtC-Ct

U]

÷

Thank You

Dan Schoeffner, Ph.DField Applications Scientist

[email protected]. Services 800-894-1304

Lisa Thompson Technical Sales Representative

[email protected]

qpcr applications using stratagene’s mx real-time pcr … · avierstr/principles/pcr.html pcr...

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