QPCR Applications using Stratagenes Mx Real-Time PCR Platform

Dan Schoeffner, Ph.D

Field Applications Scientist Dan.Schoeffner@Stratagene.com Tech. Services 800-894-1304

Polymerase Chain Reaction

DNA
Melt Anneal primers

Gene of interest (Amplicon) Forward and Reverse Primers

Extension/Measure

Melt Anneal

Q PCR Molecular Mechanism

Exponential amplification of the original DNA sequence (template) to create copies of part of the sequence (amplicon)

Xn=X0 (1+E) 2n n
X = DNA concentration X0= Starting DNA concentration Xn= DNA concentration at cycle n E = Efficiency of PCR reaction, 0-1

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

Influence of Reaction Efficiency

Typical PCR Amplification Plot

Fluorescence (R)

Baseline

Am p

lifi ca tio n

Raw Signal (R)

Threshold
Ct
Cycle #

Ct = Fractional PCR cycle number at which the fluorescence

intensity crosses the established threshold line.

A 1Ct difference between samples represents 2x more transcript

Why is QPCR superior to PCR

96 technical replicates

Variability using QPCR

Variability using endpoint PCR

Gel-based quantification
1 2 1 2

Real-time quantification
1 2

Unpredicable Amplification Plots with Endpoint Analysis

Chemistries used in QPCR

TET TET Alx350 Alx350 FAM FAM HEX HEX TAM TAM JOE Cy3 JOE Cy3 TxRd TxRd Cy5 Cy5 ROX ROX

350nm

700nm

Fluorescence Detection
Light
n io t p or s Ab n io s is Em

Light

Quantitative PCR Chemistries

dsDNA Binding Probe Based Detection

SYBR Green TaqMan Molecular Beacons Lux primers Hybridization probes ScorpionsTM Amplifluor probes FRET

Chemistries
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 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 <2C 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 green
Raw Fluorescence [R]

SYBR Green I Thermal Profile

Negative First Derivative [-R(T)]

Activation Amplification

Dissociation

Chemistries
Taqman probes
R

Pro:
Q Sequence specific Possibility to do multiplex - have GOI and normalizer in the same well, doing comparative quantification Q

Taq

Con:
R Q
Taq

More difficult to design Expensive

Linear Taqman Probe Design

Probe Tm 5-10C 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 Taqman Probe Modifications

Increase thermal duplex stability Improve specificity Raise Tm by up to 8C per LNA Allow shorter probe design (~13bp)
(www.proligo.com) O O O

DNA
Base
O

LNA
Base
O O O-

O P OO

O P

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

Introduction to MxPro software Critical Setting Threshold Baseline

Analysis term settings Algorithms

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 Value
Separates the data from the noise. Valid for Ct calculations if placed during exponential amplification. 3 Options: Amplification based threshold (Minimizes variability between replicates) 10 times the noise during early cycles. Manual (click and drag)
On exponential phase. Lines are parallel. Minimize variability

Threshold- Amplification Based

Baseline Subtraction
35000 30000 25000 20000 15000 10000 5000 0 -5000 0 10 20 Cycle # 30 40 R

R R
1

dR dR
1

Assay Development and Validation

Xn=X0 (1+E)

Q-PCR Assay Design Considerations

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

Q-PCR Assay Process

Experimental Design Oligo Design

Design Synthesis

Test oligos
Gel
Primers

Optimization Validation Run and Analyze

Probe Enzyme, dNTP, Mg++

Experimental Design
Replicates n
Biological
Depends on biological variability (CV/Power Analysis)

Technical(qPCR) Independent experiments

Reflects experimental error (n=3 is sufficient)

Ensures biological relevance (n=2 is sufficient)

Concordance of Results?

General Strategy for New QPCR Assay 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 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 - Testing Oligos

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 Validation
Standard Curve Metrics
Serial dilution of a positive control or amplified target Standard Curve

%Eff =10[-1/slope] -1
Ct Ct
Log transform

Quantity

LogQuantity

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

Assay Validation
Standard Curve Metrics

Eff=105.6% R2=0.97

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

Standard Curve Metrics

Eff=105.0% 2 R =1.0 11.5-30.2 Cts

Primer 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 <2C (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 Optimization
Primers
What if I can not identify primers sequences in my gene of interest that have the same annealing temp?
Tm of primers depends on concentration:
perform a primer matrix test to identify optimal concentration using SYBR chemistry
50 nM 50 nM 100 nM 150 nM 300 nM 600 nM 100 nM 150 nM 300 nM 600 nM

SYBR based

Assay Optimization
Primers
Aims:
low Ct values sensitivity no unspecific amplification or primer dimers specificity Low interreplicate variability high efficiency of amplification
100/150 150/100 NTC 100/150 NTC positive controls Ct = 3 150/100 NTCs

Primer titration 50 nM 200 nM duplicates for pos. Control & NTC

QPCR 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 downstream analysis. Justification for omitting data or re-assay

QPCR 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 Controls

Passive 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 Controls

Passive Reference Fluor- Example
Signal uniformity across 96 replicate wells

QPCR Assay Controls

Passive Reference Fluor- Example
~8% CV of Raw (R) Rox Signal across 96 wells

QPCR Assay Controls

Passive Reference Fluor- Example
~8% CV of Raw (R) Fam Signal across 96 wells

QPCR Assay Controls

Passive Reference Fluor- Example
<1% CV of Ct for Rox normalized, baseline subtracted (dRn) across 96 wells

QPCR Assay Controls

Positive 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 Controls

Positive 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

QPCR Assay Controls

Negative QPCR Controls 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 Control Specificity

Negative QPCR Control
Deriviation of fluorescence (RT) Standard Melting Temp. (Tm)

QPCR Assay Control Specificity

Negative QPCR Control

Sample

NTC, NoRT

QPCR Assay Control Specificity

Negative QPCR Control

Standard

QPCR Assay Control Specificity

Negative QPCR Control

Primer dimers NTC

BAD !

QPCR Assay Control Specificity

Negative QPCR Control

Standard

QPCR Assay Control Specificity

Negative QPCR Control

BAD !

Primer dimers
NTC

gDNA NoRT

QPCR Assay Control

Summary
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 Listserver qpcrlistserver@yahoogroups.com Contact Stratagene Technical Services (800) 894-1304, Pacific Standard Time QPCRSystemsSupport@Stratagene.com 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

Comparative Quantification
Given two samples: What is the difference in gene expression?
Control Gene of Interest Ct Unknown Ct CtGOI
(1+E) [CtC-CtU]

Normalizer

Ct

Ct

CtNorm
(1+E) [CtC-CtU]

Norm. ratio

Thank You
Dan Schoeffner, Ph.D Field Applications Scientist Dan.Schoeffner@Stratagene.com Tech. Services 800-894-1304 Lisa Thompson Technical Sales Representative Lisa.Thompson@Stratagene.com