Introduction to Real Time PCR (Q-PCR/qPCR/qrt-PCR): qPCR Technology Webinar Series Part 1

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Transcript of Introduction to Real Time PCR (Q-PCR/qPCR/qrt-PCR): qPCR Technology Webinar Series Part 1

Introduction To Real-Time Quantitative PCR (qPCR) SABiosciences, A QIAGEN Company www.sabiosciences.com

Introduction to Real-Time Quantitative PCR (qPCR)Webinar-related questions: QIAwebinars@qiagen.comTechnical Support:BRCsupport@qiagen.com

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Welcome to our four-part webinar series on qPCR2qPCR technology overview, applications, data analysis and service solutions

Part 1: Introduction to Real-Time PCR (Q-PCR / qPCR/ qrt-PCR)

Part 2: Advanced Real-Time PCR Array Technology Coding and Noncoding RNA Expression Analysis

Part 3: PCR Array Data Analysis Tutorial

Part 4: Accelerate your Discovery with QIAGEN Service Solutions for Biomarker Research

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Legal disclaimerQIAGEN products shown here are intended for molecular biology applications. These products are not intended for the diagnosis, prevention or treatment of a disease.For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.

Sample to InsightDisclaimer3

Aspects of a good real-time PCR assay4Agenda4Real-time PCR overview and applications1Steps involved in real-time PCR2Real-time PCR reporter chemistries3

Data and analysis5

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How does qPCR work?Question: How far apart are the two cars?The cars race at same speed to finish line As Car 1 crosses finish line, calculate time for Car 2 to finishCalculate the difference in starting position mathematically (distance = rate time)

Sample to InsightCopy:We will start with a brief explanation of what you actually use qPCR for any why. There are generally 2 types; relative real-time PCR, which we will discuss today, and absolute PCR.

Relative real-time PCR: You have 2 samples and you want to know what is the difference in these genes in our samples? Ex. Is MIC up/down regulated in a cancer sampleAbsolute PCR: We use a known standard and we want to know the quantity of something/ how many copies of something is present (ex. Virus in particular volume)

Experiment: how far apart are these 2 cars from their initial starting position. Is car 2 three or four times as far away from car 1? What is the difference? She knows she can develop a race with the equation d=RxT. She knows these cars are identical, which is an important aspect of qPCR. We are looking at the same gene in different samples. And if we know that the cars are moving at the same rate, then the difference in the initial distance is proportional to the difference in time. If these cars (or samples) are going at the same rate, then the distance should be proportional to how long it takes car 2 to cross the finish line. In relative real time PCR, we are doing the same thing. We are using a kinetic race of the real time PCR reaction to go through and calculate the difference in our starting analytes.

What is real time PCR? Explain what are you trying to achieve? have 2 samples (control, experimental), What is difference? Is gene expression going up or down? 2 samples, same gene, what is relative difference? (car example: scientist cannot see the cars at the different points visually, so she devises an experiment w/ rate*time to calculate initial difference eg. Car race w/ finish line to NY; first car crosses finish line, start a timer to see when second car finishes relative to the first one) qPCR machines do the same, kinetic race. Calculates difference between analytes.

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How does qPCR work?

Question: How far apart are the two cars?Many cars how to differentiate cars of interest

Sample to InsightCopy:Our scientist needs to identify only these 2 cars of interest from all of the other cars out there. If you think about qPCR, you are doing a kinetic race between our 2 samples. Our finish line is the threshold line in our instrument, and instead of a car engine we have the kinetics of our qPCR reaction, and instead of the distance and time, we are talking about the difference in Ct values.

(idealized reaction) Actually many cars, needs to differentiate the cars of interest between all of the other cars (specificity). Also, what if one car is lost and unable to see the car (sensitivity; affects data analysis). If one car goes off path (to Canada), or car falls apart and rate changes (efficiency; affects data analysis)? These reactions needs to be efficient (as close to 100% amplification efficiency as possible), specific (each assay to recognize only 1 target), and sensitive (need to recognize an optimal amount of copies, usually around 20 copies).6

How does qPCR work?Question: How far apart are the two cars?The cars race at same speed to finish line As Car 1 crosses finish line, calculate time for Car 2 to finishCalculate difference in starting position mathematically (distance = rate time)

Sample to InsightqPCR is the engine to race to finish line (threshold). Amplify gene to threshold. As 1 samples crosses threshold, watch other anaylte. Mathematically solve. Look at differences in cycles it takes for 2nd samples to cross finish line (not really time). Look at different starting amounts. qPCR is a kinetic race (specificity, sensitivity, amplification efficiency). Look at difference in Ct value.

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Seminar topicsWhat is qPCR? Applications and workflowqPCR for gene expression: What is the change in gene expression during differentiation?Factors critical for a successful qPCR assay RNA purity and integrityReverse transcription qPCR in actionReporter chemistries Characteristics of a good qPCR assayAnalyzing qPCR curvesData and analysis

Sample to InsightGene expression plus more! (methylation, quantify viruses or bacteria)Example (pluripotent stem cells experiment)Influencing factors, Factors influencing the performance of a qPCR assayRNA quality (purity/ integrity)RT reaction- critical to make cDNA we need, if compromised it will determine cDNA and affect our sensitivity, if not good cDNA you cannot detect gene of interestqPCR diagram, what is actually happening in your CR tube and how do you go from a regular end-point PCR reaction to a Real-time quantitative PCR reaction.Well talk about how to measure the fluorescence to make this reaction real-time and the main products to use to measure thiss, SYBR green, hydrolysis probes, when to use whichCharacteristics of GOOD real time PCR assay (validation)Analyzing qPCR curvesSome analysis8

What is qPCR? Applications and workflowWhat does real-time qPCR stand for?Quantitative polymerase chain reaction (qPCR) is a sensitive and reliable method for detection and quantification of nucleic acid (DNA and RNA) levels.

It is based on detection and quantification of fluorescence emitted from a reporter molecule in real time.

Detection occurs during the accumulation of the PCR product with each cycle of amplification. This allows for monitoring of the PCR reaction during the early and exponential phases where the first significant increase in the amount of PCR product correlates to the initial amount of target template.

Sample to Insight(read) gold standard to look at CNV, RNA (mRNA, miRNA, lncRNA (just need a good cDNA copy). It is usually used to validate other methods for gene expression. It is reliable: once you have an optimized assay, its very reliable reactionIt is sensitive: we can see down to a single copy if we use digital PCR, typically, if we have 20 copies in a reaction we can see that with a good primer design.(read) have 1 component for thermocycling block component, 1 for exciting molecule and detecting fluorescence; cannot measure individual reaction, but can measure amount of fluorescence, proportional to amount of product made each cycle (end-point is just the amount at the end of a set number of cycles) qPCR has a dynamic linear range, quantitate copies ( 20 to 20*10^7) without dilutions (ELISA, western blots) and gels(read) measure the reaction at the right time. Set threshold where we have non-limiting reaction (exponential phase) Look at curves at linear and log scale. Allows good data from reaction. No rate-limiting steps, then in theory the reaction is moving at 100% . Low threshold could be background noise, high threshold

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What is qPCR? Applications and workflowApplications for qPCR

Gene expression profiling analysismiRNA expression profiling analysis

SNP genotyping and allelic discriminationSomatic mutation analysisCopy number detection / variation analysisChromatin IP quantificationDNA methylation detection Pathogen detectionViral quantification

Sample to InsightCan be very powerful research/ diagnostic instrument

RNA routine; start with RNA, convert this to cDNA and detect whatever expression we want to look at

DNA SNP Genotyping & allelic discrimination (read)Somatic Mutation Analysis- individual mutation, insertions, deletionsCopy Number Detection/Variation Analysis- copy number of individual genes or lociChromatin IP Quantification- Chromatin Immunoprecipitation experiment, which is a powerful method to identify where protein and DNA are binding together on regions on gDNA, also for understanding histone code, insight into insight to confirmation of chromatin structure (epigenetic mechinisms)DNA Methylation Detection- gNDA to Bisulfite conversion to methyl-sensitive PCR or restriction enzymes to cleaev DNA based on methylation paterns and identify that with PCRPathogen Detection can use relative, or use absolute and incorporate standard, which we know how many copies we have so that we know the stoiceometry of what were trying to measure.Viral Quantification -

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