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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
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Polymerase Polymerase Chain Chain
ReactionReactionMeltAnneal primers
MeltAnneal
+
Extension/Measure
Gene of interest (Amplicon)
DNADNA
Forward and Reverse Primers
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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
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Influence of Reaction EfficiencyInfluence of Reaction Efficiency
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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
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Why is QPCR superior to PCRWhy is QPCR superior to PCR
96 technical replicates
Variability using endpoint PCR
Variability using QPCR
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GelGel--based quantificationbased quantification
12
21
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RealReal--time quantificationtime quantification
12
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UnpredicableUnpredicable Amplification Plots with Amplification Plots with Endpoint AnalysisEndpoint Analysis
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Chemistries used in QPCRChemistries used in QPCR
Alx350Alx350Alx350 FAMFAMFAM
TETTETTET HEXHEXHEX
ROXROXROX Cy5Cy5Cy5Cy3Cy3Cy3
TAMTAMTAM TxRdTxRdTxRd
JOEJOEJOE
350nm 700nm
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Absorption
Lightλ
Emission
Lightλ
Fluorescence DetectionFluorescence Detection
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Quantitative PCR ChemistriesQuantitative PCR Chemistries
SYBR Green
TaqMan®Molecular BeaconsLux® primersHybridization probesScorpionsTMAmplifluor® probes FRET
dsDNA Binding
Probe Based Detection
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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
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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)
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SYBR greenSYBR green
SYBR Green ISYBR Green I™™ Thermal ProfileThermal Profile
Activation Amplification Dissociation
Raw Fluorescence [R]
Negative First Derivative [-R’(T)]
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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
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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
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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
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Introduction to Introduction to MxProMxPro softwaresoftware
Critical Setting
•Threshold •Baseline
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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
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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
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ThresholdThreshold-- Amplification BasedAmplification Based
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Baseline SubtractionBaseline Subtraction
-5000
0
5000
10000
15000
20000
25000
30000
35000
0 10 20 30 40
Cycle #
R
R
dR
dR1
R1
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Xn=X0 (1+E)n
Assay Development and Assay Development and ValidationValidation
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• 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
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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
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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?
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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
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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)
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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)
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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
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Assay ValidationAssay ValidationStandard Curve Metrics
Eff=105.6%R2=0.97
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Assay ValidationAssay ValidationStandard Curve Metrics
Eff=105.0%R2=1.011.5-30.2 Cts
Copies
Select the best performing and most appropriate range for samples to be run
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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)
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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?
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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
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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
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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
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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
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QPCR Assay ControlsQPCR Assay ControlsPassive Reference FluorPassive Reference Fluor-- ExampleExample
Signal uniformity across 96 replicate wells
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QPCR Assay ControlsQPCR Assay ControlsPassive Reference FluorPassive Reference Fluor-- ExampleExample
~8% CV of Raw (R) Rox Signal across 96 wells
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QPCR Assay ControlsQPCR Assay ControlsPassive Reference FluorPassive Reference Fluor-- ExampleExample
~8% CV of Raw (R) Fam Signal across 96 wells
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QPCR Assay ControlsQPCR Assay ControlsPassive Reference FluorPassive Reference Fluor-- ExampleExample
<1% CV of Ct for Roxnormalized, baselinesubtracted (dRn) across 96 wells
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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
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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
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• 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
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QPCR Assay Control SpecificityQPCR Assay Control SpecificityNegative QPCR ControlNegative QPCR Control
Standard
Deriviation of fluorescence (R’T)Melting Temp. (Tm)
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QPCR Assay Control SpecificityQPCR Assay Control SpecificityNegative QPCR ControlNegative QPCR Control
Sample
NTC, NoRT
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Standard
QPCR Assay Control SpecificityQPCR Assay Control SpecificityNegative QPCR ControlNegative QPCR Control
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Primer dimers
NTC
BAD !
QPCR Assay Control Specificity QPCR Assay Control Specificity Negative QPCR ControlNegative QPCR Control
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Standard
QPCR Assay Control Specificity QPCR Assay Control Specificity Negative QPCR ControlNegative QPCR Control
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QPCR Assay Control Specificity QPCR Assay Control Specificity Negative QPCR ControlNegative QPCR Control
NTC
NoRT
BAD !
gDNAPrimerdimers
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• 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
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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
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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]
÷
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Thank You
Dan Schoeffner, Ph.DField Applications Scientist
[email protected]. Services 800-894-1304
Lisa Thompson Technical Sales Representative