Introduction To Quantitative PCR - Agilent

Introduction to Quantitative PCRMethods and Applications Guide

Introduction to Quantitative PCRMethods and Applications GuideIN 70200 DUS and CanadaOrders: 800-227-9770 x3Technical Service: 800-227-9770 x2For a list of worldwide distributors, please visit /genomics Agilent Technologies, Inc. 2012

Table of ContentsTable of Contents . 2Introduction to Quantitative PCR . 1Real-Time vs. Endpoint Quantitative PCR . 2Experimental Design . 5Methods of Quantification . 6Standard Curve . 6Relative Quantification . 8QPCR Chemistry Options . 9Primer and Probe Design . 16Dye and Quencher Choice . 20QPCR Reagent Choice . 21Probe and Primer Synthesis . 24Reference Dye Considerations . 24Nucleic Acid Sample Preparation. 26DNA . 26RNA. 26Total RNA vs. mRNA . 26Measuring RNA Quality . 27Storage of RNA . 29Using Cell Lysates in Real-Time QPCR . 30Reverse Transcription Considerations . 32Converting RNA to cDNA with the AffinityScript QPCR cDNA SynthesisKit . 32Reverse Transcription Priming . 32Oligo(dT) Priming. 32Random Priming . 33Combined Oligo(dT)/Random Priming . 34Gene-Specific Priming . 34QRT-PCR Reactions: One-step vs. Two-step . 35One-step . 35Two-step . 36Controls for Quantitative PCR Experiments. 38Positive Controls . 38Negative Controls . 41Passive Reference Dye . 42Approaches to Normalizing Gene Expression . 43Variability in Starting Cell Number . 43Variability in the Reverse Transcription Reaction . 44Sample Normalization using Reference Genes . 44Assay Optimization . 47Primer Optimization Guidelines. 47Primer Optimization with SYBR Green I. 48Primer Optimization with Fluorescent Probes . 52Probe Concentration Optimization Guidelines . 52Probe Optimization Data Analysis. 52Standard Curves for Analysis of QPCR Assay Performance . 53PCR Reaction Efficiency . 54Precision. 54Sensitivity . 54Standard Curve Examples . 55Further Optimization . 56Multiplex Assay Considerations. 56The Ideal Assay . 58QPCR Experiment Data Analysis . 59Ensuring Your Ct Values are Accurate . 59Raw Fluorescence Values . 59Setting the Baseline. 60

Setting the Threshold . 63Dissociation Curves (Only for SYBR Green I) . 65Controls . 66Replicate Agreement. 67Standard Curve Quantification . 67Relative or Comparative Quantification . 69 Ct Method . 69Efficiency-corrected Comparative Quantitation . 70Comparative Quantitation Module in the MxPro Software. 71Qualitative PCR . 80Other Applications of Plate Reads and Endpoint QPCR . 81Multiple Experiment Analysis . 83Applications. 83Considerations for Multiple Experiment Analysis . 83Threshold Determination in Multiple Experiment Analysis . 84Multiple Experiment Analysis Applications: How-To Examples . 88Application 1: How to Find the Initial Template Quantity in anUnknown Sample Using Standards from a Separate Experiment . 88Application 2: How to Normalize GOI Data to an Assay from aSeparate Experiment . 90Appendix . 93Primer Optimization Reaction Example . 93Probe Optimization Reaction Example . 95References. 96Fast Track Education Program . 97QPCR Glossary . 98Experiment and Chemistry Terms. 98Sample- and Well-Type Terms . 100Analysis Terms. 101Fluorescence Reading Terms . 103QPCR References and Useful Websites . 105QPCR References . 105Useful Websites . 105Product Listings & Ordering Information . 107Endnotes . 108

Introduction to Quantitative PCRWhether you are a novice or experienced user, our goal is toensure that you are running quantitative PCR (QPCR) experimentsquickly, efficiently, and affordably. Our Mx family of QPCRSystems, MxPro QPCR Software, premiere QPCR Systems ServiceProgram, complete line of QPCR and QRT- PCR reagents, and FastTrack QPCR Education Program is the total package for yourQPCR research. At Agilent Technologies, we are committed toproviding you with the most comprehensive and easy- to- usesupport programs. The Introduction to Quantitative PCR Methodsand Applications Guide was written by our Field ApplicationsScientists and Technical Services Department in order to ensurethat you are provided with the start- up support necessary to beginusing your QPCR instrument, as well as an explanation of thetheoretical basis for the materials used in QPCR techniques. Thisguide is also designed for more experienced scientists, who willfind clear guidelines for data analysis and interpretation of resultsto ensure better quality experimental results.You will find that Introduction to Quantitative PCR provides clearsteps for learning the details of QPCR methods, how to use thesemethods effectively, and the most appropriate analysis techniquesto provide reliable and reproducible results. The guide starts witha brief introduction to QPCR and experimental design. This isperhaps the most crucial step in the QPCR process as it lays thegroundwork for every other aspect of the assay. The guide thendiscusses important experimental design specifics such as primerand probe design, dye and reagent choice, assay optimization, anddata analysis. Detailed discussions on Sample Preparation, ReverseTranscription Considerations, Gene Expression Normalization andMultiple Experiment Analysis are also included.1

Real-Time vs. Endpoint Quantitative PCRPCR technology is widely used to aid in quantifying DNA becausethe amplification of the target sequence allows for greatersensitivity of detection than could otherwise be achieved. In anoptimized reaction, the target quantity will approximately doubleduring each amplification cycle. In quantitative PCR (QPCR), theamount of amplified product is linked to fluorescence intensityusing a fluorescent reporter molecule. The point at which thefluorescent signal is measured in order to calculate the initialtemplate quantity can either be at the end of the reaction(endpoint semi- quantitative PCR) or while the amplification is stillprogressing (real- time QPCR).In endpoint semi- quantitative PCR, fluorescence data are collectedafter the amplification reaction has been completed, usually after30–40 cycles, and this final fluorescence is used to back- calculatethe amount of template present prior to PCR. This method ofquantification can give somewhat inconsistent results, however,because the PCR reaction efficiency can decrease during lateramplification cycles as reagents are consumed and inhibitors to thereaction accumulate. These effects can vary from sample to sample,which will result in differences in final fluorescence values thatare not related to the starting template concentrations. As shownin Figure 1, the data collected at the reaction endpoint are notuniform even when identical samples are being amplified.The data spread of endpoint values demonstrates that datameasured following amplification are not uniform or reproducibleenough to be useful for the precise measurements required forquantitative analysis. For applications that do not require a greatdeal of copy number discrimination, such as qualitative studieswhich just seek to determine whether or not a target sequence ofinterest is present or not, end- point measurements are generallysufficient.The more sensitive and reproducible method of real- time QPCRmeasures the fluorescence at each cycle as the amplificationprogresses. This allows quantification of the template to be basedon the fluorescence signal during the exponential phase ofamplification, before limiting reagents, accumulation of inhibitors,or inactivation of the polymerase have started to have an effect onthe efficiency of amplification. Fluorescence readings at theseearlier cycles of the reaction will measure the amplified templatequantity where the reaction is much more reproducible fromsample to sample than at the endpoint.2

Figure 1QPCR run with 96 identical reactions. Note that the PCR reaction endpoint variation (i.e. 40 cycles) ismuch greater as the reaction progresses.In real- time QPCR, a fluorescent reporter molecule (such as adouble- stranded DNA- binding dye or a dye- labeled probe) is usedto monitor the progress of the amplification reaction. With eachamplification cycle, the increase in fluorescence intensity isproportional to the increase in amplicon concentration, with theQPCR instrument system collecting data for each sample duringeach PCR cycle. The resulting plots of fluorescence vs. cyclenumber for all the samples are then set with their backgroundfluorescence at a common starting point (a process known asbaseline correction). Then, a threshold level of fluorescence is setabove the background but still within the linear phase ofamplification for all the plots. The cycle number at which anamplification plot crosses this threshold fluorescence level is calledthe “Ct” or threshold cycle. This Ct value can be directlycorrelated to the starting target concentration of the sample. Thegreater the amount of initial DNA template in the sample, theearlier the Ct value for that sample (Figure 2). The MxPro analysissoftware determines the Ct value for each sample, based on certainuser- defined parameters. If a standard curve dilution series hasbeen run on the same plate as the unknown samples, the softwarewill compare the Ct values of the unknown samples to thestandard curve to determine the starting concentration of eachunknown. Alternatively, the software can use the Ct values togenerate relative comparisons of the change in templateconcentration among different samples.3

Figure 2Principles of real-time fluorescence detection and QPCR target concentration measurements usingthreshold cycle (Ct). The Ct is inversely proportional to the initial copy number. Only when the DNAconcentration has reached the fluorescence detection threshold can the concentration be reliablyinferred from the fluorescence intensity. A higher initial copy number will correlate to a lower thresholdcycle.Real- time quantitative PCR is being used in a growing number ofresearch applications including gene expression quantification,expression profiling, single nucleotide polymorphism (SNP) analysisand allele discrimination, validation of microarray data, geneticallymodified organisms (GMO) testing, monitoring of viral load andother pathogen- detection applications.4

Experimental DesignThe core idea that will guide the development of yourexperimental design is: “What is the fundamental scientificquestion that you are trying to answer?” For each project, thereare a number of considerations that need to be addressed: What is the goal of the experiment?What questions are to be answered?What is the system being studied?What is the total number of genes to be analyzed?What control samples (calibrators) and genes (normalizers orreference genes) will be used to measure the changes inexpression levels?What is the source of the target sequence?Are there any limitations to the amount of target materialavailable?What is the sensitivity required to obtain the data necessary toanswer the experiment's fundamental question?The answers to these questions will determine which QPCRapproach is best suited to the requirements and objectives of theexperiment (Figure 3).Figure 3Flowchart showing a typical experimental design process based on the goals and requirements of theassay.5