QPCR Applications using Stratagene s Mx Real-Time PCR Platform

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1 QPCR Applications using Stratagene s Mx Real-Time PCR Platform Dan Schoeffner, Ph.D Field Applications Scientist Tech. Services

2 Polymerase Chain Reaction Melt Anneal primers Extension/Measure DNA Gene of interest (Amplicon) Forward and Reverse Primers Melt Anneal +

3 QPCR Molecular Mechanism Exponential amplification of the original DNA sequence (template) to create copies of part of the sequence (amplicon) X n =X 0 (1+E) n 2 n X = DNA concentration X 0 = Starting DNA concentration X n = DNA concentration at cycle n E = Efficiency of PCR reaction, 0-1

4 Influence of Reaction Efficiency

5 Typical PCR Amplification Plot Fluorescence (R) Baseline Amplification Raw Signal (R) Threshold Ct Cycle # C t = Fractional PCR cycle number at which the fluorescence intensity crosses the established threshold line. A 1C t difference between samples represents 2x more transcript

6 Why is QPCR superior to PCR 96 technical replicates Variability using QPCR Variability using endpoint PCR

7 Gel-based quantification

8 Real-time quantification 1 2

9 Unpredicable Amplification Plots with Endpoint Analysis

10 Chemistries used in QPCR TET HEX TAM TxRd Alx350 FAM JOE Cy3 ROX Cy5 350nm 700nm

11 Fluorescence Detection Light λ Light λ Absorption Emission

12 Quantitative PCR Chemistries dsdna Binding Probe Based Detection SYBR Green TaqMan Molecular Beacons Lux primers Hybridization probes ScorpionsTM Amplifluor probes FRET

13 Chemistries SYBR green dsdna binding dyes 1000x increase in fluorescence 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

14 Primer 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)

15 SYBR green Raw Fluorescence [R] SYBR Green I I Thermal Profile Negative First Derivative [-R (T)] Activation Amplification Dissociation

16 Chemistries Taqman probes Taq R Q R Q Pro: Sequence specific Possibility to do multiplex - have GOI and normalizer in the same well, doing comparative quantification Taq R Q Con: More difficult to design Expensive

17 Linear Taqman Probe Design Probe T m 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

18 Linear Taqman Probe Modifications Increase thermal duplex stability DNA LNA Improve specificity Raise T m by up to 8 C per LNA O O Base O O Base Allow shorter probe design (~13bp) O O O (www.proligo.com) O P O - O P O - 2'-O, 4'-C methylene bridge locks conformation

19 Introduction to MxPro software Critical Setting Threshold Baseline

20 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

21 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 Value

22 Threshold- Amplification Based

23 Baseline Subtraction R R R 1 dr dr Cycle #

24 Assay Development and Validation Xn=X0 (1+E) n

25 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

26 Q-PCR Assay Process Experimental Design Oligo Design Design Synthesis Gel Test oligos Optimization Validation Primers Probe Enzyme, dntp, Mg ++ Run and Analyze

27 Experimental Design Replicates n Depends on biological variability Biological (CV/Power Analysis) Reflects experimental error Technical(qPCR) (n=3 is sufficient) Independent experiments Ensures biological relevance (n=2 is sufficient) Concordance of Results?

28 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

29 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)

30 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)

31 Serial dilution of a positive control or amplified target Assay Validation Standard Curve Metrics Standard Curve %Eff =10 [-1/slope] -1 Ct Log transform Ct Quantity LogQuantity Expect: High efficiency (E = %) Good linear fit (R 2 > 0.98) 3-5 logs of dynamic range ~1 % CV variation among triplicates

32 Assay Validation Standard Curve Metrics Eff=105.6% R 2 =0.97

33 Assay Validation Standard Curve Metrics Select the best performing and most appropriate range for samples to be run Copies Eff=105.0% R 2 = Cts

34 Primer 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: , 200bp ; Taqman , 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)

35 Assay Optimization Primers What if I can not identify primers sequences in my gene of interest that have the same annealing temp? T m of primers depends on concentration: perform a primer matrix test to identify optimal concentration using SYBR chemistry SYBR based 50 nm 100 nm 150 nm 300 nm 600 nm 50 nm 100 nm 150 nm 300 nm 600 nm

36 Assay Optimization Primers Aims: low Ct values sensitivity Primer titration 50 nm 200 nm duplicates for pos. Control & NTC no unspecific amplification or primer dimers specificity 150/100 NTC positive controls Ct = 3 100/ /100 NTCs Low interreplicate variability 100/150 NTC high efficiency of amplification

37 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

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

39 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

40 QPCR Assay Controls Passive Reference Fluor- Example Signal uniformity across 96 replicate wells

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

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

43 QPCR Assay Controls Passive Reference Fluor- Example <1% CV of Ct for Rox normalized, baseline subtracted (drn) across 96 wells

44 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

45 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

46 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

47 QPCR Assay Control Specificity Negative QPCR Control Deriviation of fluorescence (R T) Standard Melting Temp. (Tm)

48 QPCR Assay Control Specificity Negative QPCR Control Sample NTC, NoRT

49 QPCR Assay Control Specificity Negative QPCR Control Standard

50 QPCR Assay Control Specificity Negative QPCR Control Primer dimers BAD! NTC

51 QPCR Assay Control Specificity Negative QPCR Control Standard

52 QPCR Assay Control Specificity Negative QPCR Control BAD! Primer dimers NTC gdna NoRT

53 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

54 QPCR Listserver Contact Stratagene Technical Services (800) , Pacific Standard Time Webinars and Introduction to QPCR Guide: 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

55 Comparative Quantification Given two samples: What is the difference in gene expression? Control Unknown Gene of Interest Ct Ct Ct GOI (1+E) [Ct C -Ct U ] Normalizer Ct Ct Ct Norm (1+E) [Ct C -Ct U ] Norm. ratio

56 Thank You Dan Schoeffner, Ph.D Field Applications Scientist Tech. Services Lisa Thompson Technical Sales Representative

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