Introduction To Real Time Quantitative PCR (qpcr)

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1 Introduction To Real Time Quantitative PCR (qpcr) SABiosciences, A QIAGEN Company

2 The Seminar Topics The advantages of qpcr versus conventional PCR Work flow & applications Factors influencing the performance of a qpcr assay Reporter chemistry Terms commonly used Ct method for relative quantifications How to set up a qpcr reaction Data output & analysis -2-

3 What Real-Time qpcr Stands for? Real time qpcr is the most sensitive and reliable method for detection and quantification of nucleic acids (DNA, cdna, & RNA) levels. It is based on detection and quantification of fluorescence emitted from a reporter molecule at real time. This detection occurs during the accumulation of the PCR product with each cycle of amplification, thus allows monitoring the PCR reaction during early & exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target template. -3-

4 Understanding Reaction Kinetics in PCR Theoretically, the PCR reaction doubles the DNA amount after completing each cycle exponential reaction! The reaction, however, finally tails off and reaches a plateau -4-

5 Understanding Kinetics in PCR Amplification Plot (Linear scale) Plateau End point data collection at plateau (gel analysis) Fluorescence Signal Reactions start varying due to reagent depletion & decreased PCR efficiencies (enzyme activity, More product competing for primer annealing Real time PCR does early phase detection at the exponential state Precisely proportional to input amounts -5-

6 Real-Time qpcr vs End Point Detection (cont.) Look different Look similar The RNA was reversed transcribed to cdna followed by real time or end point analysis by gel electrophoresis. (A) same starting amounts of RNA; (B) Different starting amounts. Y, in linear scale; M: DNA marker. -6-

7 Wide Dynamic Detection Range DEFINITION: The range of initial template concentrations over which accurate measurement can be achieved HIGX1A Gene NTC At least 7 log difference range can be detected to accommodate varying initial sample input amounts/gene expression levels, in contrast to <2 log difference by traditional PCR (End point analysis by gel electrophoresis) -7-

8 Wide Range of Applications RNA DNA Gene Expression Profiling Analysis Microarray Validation mirna Expression Profiling Analysis Gene Regulation Genetic & Epigenetic SNP Genotyping & allelic discrimination Somatic Mutation Analysis Copy Number Detection/Variation Analysis Pathogen Detection Viral Quantification -8-

9 Summary of The Advantages Detect the reporter signals at early & exponential phase, in real time! Precisely detect the amplicon during each cycle proportional to the initial template amounts. Quantitative! Increased dynamic range of detection wide range of applications Increased sensitivity: detect a difference as small as 2 fold change No post PCR processing necessitated: increased assay throughput & reduced risk of carry over contaminations -9-

10 Work A Brief Look RNA (total, mrna, small RNA) Reverse transcription cdna Samples DNA Sample quality control SYBR or Probe Assay Design Assay Optimization Real Time PCR Set Up Instrument Set up & thermal cycling Data Output & Analysis -10-

11 Factors Critical For A Successful Assay DNA or RNA sample preparation Template quality Choose appropriate sample preparation kits/reagents Reverse transcription for converting RNA to cdna Template Quality Choose RT reaction kits/reagents Assay design: chemistry, specificity, PCR efficiency, & throughput & cost Choose thoroughly validated assay Running PCR Choose high quality reagents (primer, probe, master mix) Data analysis tool User friendly & streamlined data analysis module -11-

12 qpcr Components & Steps: Overview A. Templates B. Primers/Probes copies of nucleic acids 100 pg to 1 g DNA or cdna/rna Reverse Transcription Two step qpcr: (1) RT (2) qpcr C. Master Mix DNA Polymerase Mg++ dntp Buffer *Passive reference dye One step qpcr: one tube reaction Denaturation Annealing Extension Denaturation Annealing/Extension -12-

13 Real-Time qpcr Fluorescence Chemistry DNA binding agents SYBR I Dye Two most commonly used chemistries in qpcr community Hydrolysis probes Taqman probe Others, such as hybridization probes -13-

14 SYBR Green I Assay: Fluorescent DNA Binding Dye Non fluorescent SYBR I SYBR I binds to double strand DNA but not single strand DNA. Little fluorescence emitted from SYBR I in solution. SYBR I upon binding to double strand DNA emits fluorescence very brightly Simple & cost saving Fluorescent SYBR I The SYBR I signal intensities correlate with DNA amplified (amplicon amount) thus the initial sample input amounts High Specificity Is Required when using SYBR Green since SYBR I binds all double strand DNA (non specific or primer dimmer). -14-

15 Hydrolysis Based Probe ---TaqmanProbe Assay The fluorescence of the reporter dye is suppressed by the quencher Primer binding followed by extension Probe cleavage by Taq to free the reporter dye thus the fluorescence intensity correlates with the initial sample input amounts. Taq has 5 3 exonuclease activity Each amplicon needs a sequence specific probe (cost & time) -15-

16 Thermal Cycling Programs 1 Instrument default melt curve program Melt curve analysis (SYBR Only) Step 1 Activation Step 2 Stratagene Mxp3005p Data collection Step 3 Melt curve analysis -16-

17 Understanding Amplification Plots & Basic Terms Before nucleic acid level can be qualified, the raw data must be analyzed and baseline & threshold values set -17-

18 Understanding Amplification Plots & Basic Terms Rn: Fluorescence signal normalized with passive reference dye signal (Fluorescence/dye signal) Passive Reference Dye: An internal reference to correct well to well non PCR fluctuations. ROX is the most commonly used reference dye. Rn: Fluorescence signal with baseline subtracted (background signal subtracted) Baseline: background noise level before a significant amplification occurs (3 15 cycles) -18-

19 Understanding Amplificaiton Plot & Basic Terms Threshold: At which significant & specific amplification occurs (auto or manual set) exponential phase (linear doubling phase of amplification) above the highest baseline values. 10 x Rn standard deviations C t (Threshold cycle): The cycle number at which the fluorescence signal crosses threshold. Also called Cp (Cross point cycle) for LightCycler terminology. inversely correlates with initial template concentrations (amounts) -19-

20 How To Define/Set Up The Baseline Linear Amplification Plot Automated Baseline Option if an instrument has a adaptive baseline function Define manually: Baseline Ct (1) Use linear view of the plot (2) Set up the baseline reading from cycle #2 to the cycle that 2 cycles before the earliest visible amplification (3) Usually a baseline falls in 3 15 cycles -20-

21 How To Define Threshold Log View Amplification Plot Use log view of amplification plot Threshold should be higher than baseline (higher than the noise level) Threshold should at LOWER 1/3 or 1/2 of the linear phase of amplification Linear phase = exponential phase Different runs across samples for the same experiments should have the same threshold for comparison -21-

22 Tm & Melt Curve Analysis Tm and Melting Curve Analysis are important features of SYBR I based assay to monitor an assay s specificity. This assay is only applied to SYBR based qpcr but not Taqman probe assay because the amplicon of a Taqman assay is non fluorescent. When SYBR I is present, as a DNA fragment is heated, a sudden decrease of fluorescence signal is observed at Tm when 50% of molecules become single stranded thus of the loss of binding of SYBR I. Every double strand DNA molecule has a melting point Tm at which 50% DNA are single stranded. Tm depends on length, sequence, and GC content A Tm value and single melt curve peak (plotted as 1 st negative derivative df/dt) is important for assay specificity validation. -22-

23 Melt Curve Analysis: The General Program Steps Rapid heating of amplified samples to 94 C to denature the DNA Cooling the sample to 60 C to let DNA double strands anneal Slowly heating (by increasing the temperature, usually 0.2 C/sec) the sample while plotting the fluorescent signal versus temperature. As the temperature increases, and DNA melts, the fluorescent signal should decrease. There will be a significant drop of the signal when 50% DNA melts. -23-

24 Melting Curve Analysis --- Normalized Reporter Plot Plot Normalized Reporter (Fluorescence/Passive dye signal) Normalized Fluorescence Signal Rn 50% fluorescence drop Samples Tm Gene A Gene B Tm: A Tm: B Temperature -24-

25 Melt Curve Analysis st Negative Derivative Plot Plot 1 st negative Derivative Reporter delta F/delta T (the change rate) Gene A Tm: A Gene B Tm: B Single melt curve of each amplicon is required for specificity validation! Temperature -25-

26 Quantification Absolute Quantification Typical standard curves showing determination of concentration of sample of interest Goal: absolute/exact concentration of a sample is desired. The known standard & an unknown target should have very similar characteristics: *Amplicon & adjacent sequences *Primer binding sites *PCR efficiencies One standard curve One GOI & At least 5 serial dilution points needed for the known standard Limited Throughput Often applied to assays of single to a small number of genes (For example, viral load and copy number counting) -26-

27 Comparative Ct Method Quantification Gene Expression Profiling Analysis Focused on Changes in Gene Expression Across Samples Any changes upon treatment? Gene of interest A in untreated cells Ct untreated GOI A in drug treated cells Ct treated by relative measurement A Fold Change of Expression = 2 (Up or down regulation) = 2 delta (Ct treated Ct untreated) Ct (Treated Untreated) -27-

28 Reference Genes (Housekeeping Genes) For Normalization Any changes? Gene of interest A in untreated cells GOI A in drug treated cells Reference Gene B in untreated cells Ref Gene B in drug treated cells The expression level of a reference gene remain consistent under experimental conditions or different tissues A Reference Gene is aimed to normalize possible variations during: Sample prep & handling (e.g use the same number of cells from a start) RNA isolation (RNA quality and quantity) Reverse transcription efficiency across samples/experiments PCR reaction set up PCR reaction amplification efficiencies -28-

29 Normalized Gene Expression Level Any changes? Target Gene A in control cells Reference Gene B in control cells Target Gene A in drug treated cells Ref Gene B in drug treated cells Ct1 = Ct (Target A treated) Ct (Ref B treated) Ct2 = Ct (Target A control) Ct (Ref B control) Ct = Ct1 (treated) Ct2 (control) Normalized target gene expression level = 2 Ct -29-

30 Delta Delta Ct Method: A Look of Amplification Plots Ref GOI GAPDH GOI Ct = Ct (TNFα treat GAPDH treat ) ct (TNFα control GAPDH control ) The fold change = 2 Ct -30-

31 Commonly Used Housekeeping Genes -31-

32 How To Carry Out A qpcr Assay Primers/Probes Design/Optimize Template (cdna or gdna) Kits/reagents for Prep & RT/Quality Master Mix (2x) High Performance Thermal cycler User Manual/Calibration/Maintenance Good Experimental Techniques Accuracy & avoid risk of contamination -32-

33 Run qpcr---reaction Plate Set Up -33-

34 Run qpcr Thermal Cycling Program Set Up -34-

35 Run qpcr---results -35-

36 Single Primer Assay of Any Gene of Interest Thoroughly Bench validated PCR efficiency (robust) Specificity (single melt curve) Size verified (gel) Complete Genome Coverage Human Mouse Rat Rhesus Fruit Fly Easy to use protocol 5 min set up -36-

37 High Performance SYBR Green qpcr Analysis RT 2 Profiler PCR Arrays Pathway Focused (84 genes or more) Accurate, Sensitive, Specific & with Wide Dynamic Range Easy to Use & Designed for Routine Use -37-

38 RT 2 qpcr Array Work Flow: Simple & Accurate * cdna + Master mix + H 2 0 = Cocktail Simple set up! Control Treated * Simultaneous analysis of 84 GOIs of the same biological pathway * All primer assays have been thoroughly wet bench validated (efficiency + specificity) * Built in controls for: 5 Housekeeping genes Genomic DNA contamination control RTC: reverse transcription control PPC: positive PCR control * Free and easy to use data analysis portal * Know answers soon after the run -38-

39 A Complete Solution In One Package.Complete System RNA isolation kits, PCR Arrays, master mix, first strand kit, and free data analysis software Systematic controls to monitor sample quality, RT and PCR efficiency 3hours from RNA to data analysis.breadth of Pathway Content Profile the genes and pathways that you really care about More than 130 pathways for human, mouse, rat, fruit fly and rhesus Customizable by gene, set, or pathway.superb Real Time PCR Performance Reproducibility, Sensitivity, Specificity, Reliability.Broad Application Cancer, Immunology, Neuroscience, Stem Cells & much more -39-

40 Questions? Ask now or contact Technical Support M F, 9 AM 6 PM EST Telephone: (888) Thank You!

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