An Introduction to Real Time Pcr Qpcr Assay Design and Optimization

7/27/2019 An Introduction to Real Time Pcr Qpcr Assay Design and Optimization

1/56

QPCR Applications using StratageneQPCR Applications using Stratageness

Mx RealMx Real--Time PCR PlatformTime PCR Platform

Dan Schoeffner, Ph.DField Applications Scientist

[email protected]

Tech. Services 800-894-1304


2/56

PolymerasePolymerase

ChainChainReactionReaction

Melt

Anneal primers

Melt

Anneal

+

Extension/Measure

Gene of

interest

(Amplicon)

DNADNA

Forward and

Reverse Primers


3/56


4/56

Influence of Reaction EfficiencyInfluence of Reaction Efficiency


5/56

Cycle #

Fluoresce

nce(R)

BaselineRaw Signal (R)

CCtt = Fractional PCR cycle number at which the fluorescenceintensity crosses the established threshold line.

A 1CCtt difference between samples represents 2x2x more transcript

Ct

Threshold

Typical PCR Amplification PlotTypical PCR Amplification Plot

Amplifica

tion


6/56


7/56

GelGel--based quantificationbased quantification

1

2

21


8/56

RealReal--time quantificationtime quantification

1

2


9/56

UnpredicableUnpredicableAmplification Plots withAmplification Plots with

Endpoint AnalysisEndpoint Analysis


10/56


11/56


12/56

Quantitative PCR ChemistriesQuantitative PCR Chemistries

SYBR Green

TaqManMolecular Beacons

Lux primersHybridization probes

ScorpionsTM

Amplifluor probes

FRET

dsDNA Binding

Probe Based

Detection


13/56


14/56


15/56

SYBR greenSYBR green

SYBR Green ISYBR Green I Thermal ProfileThermal Profile

Activation Amplification Dissociation

Raw Fluorescence [R]

Negative First Derivative [-R(T)]


16/56


17/56


18/56

LinearLinearTaqmanTaqman Probe ModificationsProbe Modifications

Increase thermal duplex stability

Improve specificity

Raise Tm by up to 8C per LNA

Allow shorter probe design (~13bp)

(www.proligo.com)

2'-O, 4'-C methylene bridge locksconformation

O

BaseO

O

PO O-

O

BaseO

O O

PO O-

DNA LNA


19/56


20/56

Analysis term settingsAnalysis term settings

AlgorithmsAlgorithms

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 samemethod over time, identify trends

Easier to justify settings for validation, QA/QC

purposes


21/56

Threshold ValueThreshold Value

Separates the data from the noise.Separates the data from the noise.Valid for Ct calculations if placedValid for Ct calculations if placed

during exponential amplification.during exponential amplification.

3 Options:3 Options: Amplification basedAmplification based

threshold (Minimizesthreshold (Minimizesvariability betweenvariability betweenreplicates)replicates)

10 times the noise during10 times the noise duringearly 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


22/56

ThresholdThreshold-- Amplification BasedAmplification Based


23/56


24/56


25/56


26/56


27/56

G l St t f N QPCRGeneral Strategy for New QPCR


28/56

General Strategy for New QPCRGeneral Strategy for New QPCR

Assay DevelopmentAssay Development Plan to optimize assay using SYBR Green

chemistry SYBR melt curve will yield PCR specificity infothat probe based detection will not

Attempt to constrain assays to a commonthermal profile for convenience

Design amplicons compatible with probe

chemistry for possible future use in amultiplex QPCR format


29/56

Fi t A T ti Oli


30/56

First AssayFirst Assay -- TestingTesting 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, negativecontrols

150 to 300nM primers, ~100 ngs of template

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


31/56


32/56


33/56

P i S l tiP i S l ti


34/56

Primer SelectionPrimer Selection

Try to achieve similar Tm for all primers: Ideal ~60C.(Future multiplexing or use of Taqman assays in mind)

Forward and reverse primer should have Tm -4kcal/mol to avoid stable primer dimers

Design via software (Always use the same one):

Always perform a BLAST search with your amplicon andprimers( Specificity of the PCR)


35/56


36/56

QPCR Assay ControlsQPCR Assay Controls


37/56


Initial efforts should identify good controlmaterials to run during assay setup and

validation Establish a range of acceptable QPCR

performance data

Controls will dictate what data is good orbad and what should be included in down-stream analysis.

Justification for omitting data or re-assay


38/56


39/56



40/56

yPassive Reference FluorPassive Reference Fluor-- ExampleExample

Signal uniformity across 96 replicate wells


41/56



42/56

Passive Reference FluorPassive Reference Fluor-- ExampleExample

~8% CV of Raw (R)

Fam Signal across 96 wells


43/56


44/56


45/56

QPCR Assay ControlsQPCR Assay ControlsN ti QPCR C t lN ti QPCR C t l


46/56

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 QPCRreaction

May indicate high background

Negative QPCR ControlsNegative QPCR Controls


47/56


48/56


49/56


50/56


51/56

QPCR Assay Control SpecificityQPCR Assay Control SpecificityN ti QPCR C t lN ti QPCR C t l


52/56

Negative QPCR ControlNegative QPCR Control

NTC

NoRT

BAD !

gDNAPrimer

dimers

QPCR Assay ControlQPCR Assay Control


53/56

Assay controls are the main determinant of

data quality

Provide leverage for troubleshooting,

allows you to regain assay performancequickly

Easy to prepare, requires up-front effort,

worth the work in the long term

QPCR Assay ControlQ y

SummarySummary

QPCR Listserver


54/56

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 Stratagenes MxPro QPCR Software


55/56


56/56

Documents

An Introduction to Real Time Pcr Qpcr Assay Design and Optimization