Real-time PCR Handbook - Thermo Fisher Scientific
Real-time PCR handbook
Single-tube assays96- and 384-well plates384-well TaqMan Array cardsOpenArray platesCommonly used formats for real-time PCR.
Basics of real-time PCRExperimental designPlate preparationData analysisTroubleshootingDigital PCR123456
Basics of real-time PCR1
Basics of real-time PCR1.1Introduction21.2Overview of real-time PCR31.3Overview of real-time PCR components41.4Real-time PCR analysis technology61.5Real-time PCR fluorescence detection systems101.6Melting curve analysis141.7Passive reference dyes151.8Contamination prevention161.9Multiplex real-time PCR161.10 Internal controls and reference genes181.11 Real-time PCR instrument calibration19lifetechnologies.com11
Basics of real-time PCR1.1 IntroductionThe polymerase chain reaction (PCR) is one of the mostpowerful technologies in molecular biology. Using PCR,specific sequences within a DNA or cDNA template can becopied, or “amplified”, many thousand- to a million-foldusing sequence-specific oligonucleotides, heat-stable DNApolymerase, and thermal cycling. In traditional (endpoint)PCR, detection and quantification of the amplifiedsequence are performed at the end of the reaction afterthe last PCR cycle, and involve post-PCR analysis suchas gel electrophoresis and image analysis. In real-timequantitative PCR (qPCR), PCR product is measured at eachcycle. By monitoring reactions during the exponentialamplification phase of the reaction, users can determinethe initial quantity of the target with great precision.1PCR theoretically amplifies DNA exponentially, doublingthe number of target molecules with each amplificationcycle. When it was first developed, scientists reasoned thatthe number of cycles and the amount of PCR end-productcould be used to calculate the initial quantity of geneticmaterial by comparison with a known standard. To addressthe need for robust quantification, the technique of realtime quantitative PCR was developed. Currently, endpointPCR is used mostly to amplify specific DNA for sequencing,cloning, and use in other molecular biology techniques.In real-time PCR, the amount of DNA is measured aftereach cycle via fluorescent dyes that yield increasingfluorescent signal in direct proportion to the numberof PCR product molecules (amplicons) generated. Datacollected in the exponential phase of the reaction yieldquantitative information on the starting quantity of theamplification target. Fluorescent reporters used in realtime PCR include double-stranded DNA (dsDNA)–bindingdyes, or dye molecules attached to PCR primers or probesthat hybridize with PCR products during amplification.The change in fluorescence over the course of the reactionis measured by an instrument that combines thermalcycling with fluorescent dye scanning capability. By plottingfluorescence against the cycle number, the real-time PCRinstrument generates an amplification plot that representsthe accumulation of product over the duration of the entirePCR reaction (Figure 1).The advantages of real-time PCR include: Ability to monitor the progress of the PCRreaction as it occurs in real time Ability to precisely measure the amountof amplicon at each cycle, which allowshighly accurate quantification of the amountof starting material in samples An increased dynamic range of detection Amplification and detection occur in a singletube, eliminating post-PCR manipulationsOver the past several years, real-time PCR has becomethe leading tool for the detection and quantification of DNAor RNA. Using these techniques, you can achieve precisedetection that is accurate within a 2-fold range, with adynamic range of input material covering 6 to 8 orders ofmagnitude.Figure 1. Relative fluorescence vs. cycle number. Amplification plots are created when the fluorescent signal from each sample is plotted againstcycle number; therefore, amplification plots represent the accumulation of product over the duration of the real-time PCR experiment. The samplesused to create the plots in this figure are a dilution series of the target DNA sequence.2
Basics of real-time PCR1.2 Overview of real-time PCRThis section provides an overview of the steps involved inperforming real-time PCR. Real-time PCR is a variationof the standard PCR technique that is commonly used toquantify DNA or RNA in a sample. Using sequence-specificprimers, the number of copies of a particular DNA or RNAsequence can be determined. By measuring the amountof amplified product at each stage during the PCR cycle,quantification is possible. If a particular sequence (DNA orRNA) is abundant in the sample, amplification is observedin earlier cycles; if the sequence is scarce, amplificationis observed in later cycles. Quantification of amplifiedproduct is obtained using fluorescent probes or fluorescentDNA-binding dyes and real-time PCR instruments thatmeasure fluorescence while performing the thermalcycling needed for the PCR reaction.Two-step qRT-PCRReal-time PCR stepsOne-step qRT-PCRThere are three major steps that make up each cycle in areal-time PCR reaction. Reactions are generally run for 40cycles.1. Denaturation: High-temperature incubation is used to“melt” double-stranded DNA into single strands andloosen secondary structure in single-stranded DNA.The highest temperature that the DNA polymerasecan withstand is typically used (usually 95 C). Thedenaturation time can be increased if template GCcontent is high.2. Annealing: During annealing, complementarysequences have an opportunity to hybridize, so anappropriate temperature is used that is based on thecalculated melting temperature (Tm) of the primers(typically 5 C below the Tm of the primer).3. Extension: At 70–72 C, the activity of the DNApolymerase is optimal, and primer extension occursat rates of up to 100 bases per second. When anamplicon in real-time PCR is small, this step is oftencombined with the annealing step, using 60 C as thetemperature.Two-step quantitative reverse transcriptase PCR (qRT-PCR)starts with the reverse transcription of either total RNA orpoly(A) RNA into cDNA using a reverse transcriptase (RT).This first-strand cDNA synthesis reaction can be primedusing random primers, oligo(dT), or gene-specific primers(GSPs). To give an equal representation of all targets inreal-time PCR applications and to avoid the 3 bias ofoligo(dT) primers, many researchers use random primersor a mixture of oligo(dT) and random primers.The temperature used for cDNA synthesis dependson the RT enzyme chosen. After reverse transcription,approximately 10% of the cDNA is transferred to a separatetube for the real-time PCR reaction.1One-step qRT-PCR combines the first-strand cDNAsynthesis reaction and real-time PCR reaction in thesame tube, simplifying reaction setup and reducing thepossibility of contamination. Gene-specific primers (GSP)are required. This is because using oligo(dT) or randomprimers will generate nonspecific products in the one-stepprocedure and reduce the amount of product of interest.lifetechnologies.com3
Basics of real-time PCR1.3 Overview of real-time PCR components1This section provides an overview of the major reactioncomponents and parameters involved in real-time PCRexperiments. A more detailed discussion of specificcomponents like reporter dyes, passive reference dyes, anduracil DNA glycosylase (UDG) is provided in subsequentsections of this handbook.Magnesium concentrationDNA polymeraseGood experimental techniquePCR performance is often related to the thermostableDNA polymerase, so enzyme selection is critical tosuccess. One of the main factors affecting PCR specificityis the fact that Taq DNA polymerase has residual activityat low temperatures. Primers can anneal nonspecificallyto DNA during reaction setup, allowing the polymerase tosynthesize nonspecific product. The problem of nonspecificproducts resulting from mis-priming can be minimizedby using a “hot-start” enzyme. Using a hot-start enzymeensures that DNA polymerase is not active during reactionsetup and the initial DNA denaturation step.Reverse transcriptaseThe reverse transcriptase (RT) is as critical to the successof qRT-PCR as the DNA polymerase. It is important tochoose an RT that not only provides high yields of full-lengthcDNA, but also has good activity at high temperatures.High-temperature performance is also very importantfor denaturation of RNA with secondary structure. Inone-step qRT-PCR, an RT that retains its activity at highertemperatures allows you to use a GSP with a high meltingtemperature (Tm), increasing specificity and reducingbackground.dNTPsIt is a good idea to purchase both the dNTPs and thethermostable DNA polymerase from the same vendor,as it is not uncommon to see a loss in sensitivity of onefull threshold cycle (Ct) in experiments that employ thesereagents from separate vendors.4In real-time PCR, magnesium chloride or magnesiumsulfate is typically used at a final concentration of 3 mM.This concentration works well for most targets; however,the optimal magnesium concentration may vary between3 and 6 mM.Do not underestimate the importance of good laboratorytechnique. It is best to use dedicated equipment andsolutions for each stage of the reactions, from preparationof the template to post-PCR analysis. The use of aerosolbarrier tips and screwcap tubes can help decreasecross-contamination problems. To obtain tight data fromreplicates (ideally, triplicates), prepare a master mix thatcontains all the reaction components except sample. Theuse of a master mix reduces the number of pipetting stepsand, consequently, reduces the chances of cross-wellcontamination and other pipetting errors.TemplateUse 10 to 1,000 copies of template nucleic acid for eachreal-time PCR reaction. This is equivalent to approximately100 pg to 1 μg of genomic DNA, or cDNA generated from1 pg to 100 ng of total RNA. Excess template may also bringhigher contaminant levels that can greatly reduce PCRefficiency. Depending on the specificity of the PCR primersfor cDNA rather than genomic DNA, it may be important totreat RNA templates to reduce the chance that they containgenomic DNA contamination. One option is to treat thetemplate with DNase I.Pure, intact RNA is essential for full-length, high-qualitycDNA synthesis and may be important for accuratemRNA quantification. RNA should be devoid of anyRNase contamination, and aseptic conditions should bemaintained. Total RNA typically works well in qRT-PCR;isolation of mRNA is typically not necessary, although itmay improve the yield of specific cDNAs.
Basics of real-time PCRReal-time PCR primer designGood primer design is one of the most important parametersin real-time PCR. This is why many researchers choose topurchase TaqMan Assay products—primers and probesfor real-time PCR designed using a proven algorithm andtrusted by scientists around the world. If you choose todesign your own real-time PCR primers, keep in mind thatthe amplicon length should be approximately 50–150 bp,since longer products do not amplify as efficiently.In general, primers should be 18–24 nucleotides in length.This provides for practical annealing temperatures.Primers should be designed according to standard PCRguidelines. They should be specific for the target sequenceand be free of internal secondary structure. Primers shouldavoid stretches of homopolymer sequences (e.g., poly(dG))or repeating motifs, as these can hybridize inappropriately.Primer design softwarePrimer design software programs, such as OligoPerfect designer and Primer Express software, in addition tosequence analysis software, such as Vector NTI Software,can automatically evaluate a target sequence and designprimers for it based on the criteria previously discussed.At a minimum, using primer design software will ensurethat primers are specific for the target sequence and freeof internal secondary structure, and avoid complementaryhybridization at 3 ends within each primer and witheach other. As mentioned previously, good primer designis especially critical when using DNA-binding dyes foramplicon detection.1Primer pairs should have compatible melting temperatures(within 1 C) and contain approximately 50% GC content.Primers with high GC content can form stable imperfecthybrids. Conversely, high AT content depresses the Tm ofperfectly matched hybrids. If possible, the 3 end of theprimer should be GC rich to enhance annealing of the endthat will be extended. Analyze primer pair sequences toavoid complementarity and hybridization between primers(primer-dimers).For qRT-PCR, design primers that anneal to exons on bothsides of an intron (or span an exon/exon boundary of themRNA) to allow differentiation between amplification ofcDNA and potential contaminating genomic DNA by meltingcurve analysis. To confirm the specificity of your primers,perform a BLAST search against public databases to besure that your primers only recognize the target of interest.Optimal results may require a titration of primerconcentrations between 50 and 500 nM. A finalconcentration of 200 nM for each primer is effective formost reactions.lifetechnologies.com5
Basics of real-time PCR1.4 Real-time PCR analysis technologyThis section defines the major terms used in real-time PCR analysis.BaselineThe baseline of the real-time PCR reaction refers to thesignal level during the initial cycles of PCR, usually cycles3 to 15, in which there is little change in fluorescent signal.The low-level signal of the baseline can be equated to thebackground or the “noise” of the reaction (Figure 2). Thebaseline in real-time PCR is determined empirically foreach reaction, by user analysis or automated analysis ofthe amplification plot. The baseline should be set carefullyto allow accurate determination of the threshold cycle (Ct),defined below. The baseline determination should takeinto account enough cycles to eliminate the backgroundfound in the early cycles of amplification, but should notinclude the cycles in which the amplification signal beginsto rise above background. When comparing different realtime PCR reactions or experiments, the baseline should bedefined in the same way for each (Figure 2).1As the template amount decreases, the cycle number atwhich significant amplification is seen increases. With a10-fold dilution series, the Ct values are 3.3 cycles apart.Standard curveA dilution series of known template concentrations can beused to establish a standard curve for determining the initialstarting amount of the target template in experimentalsamples or for assessing the reaction efficiency (Figure 4).The log of each known concentration in the dilution series(x-axis) is plotted against the Ct value for that concentrationThresholdThe threshold of the real-time PCR reaction is the levelof signal that reflects a statistically significant increaseover the calculated baseline signal (Figure 2). It is setto distinguish relevant amplification signal from thebackground. Usually, real-time PCR instrument softwareautomatically sets the threshold at 10 times the standarddeviation of the fluorescence value of the baseline.However, the position of the threshold can be set at anypoint in the exponential phase of PCR.Figure 2. The baseline and threshold of a real-time PCR reaction.Figure 3. Amplification plot for a 10-fold dilution series.Ct (threshold cycle)The threshold cycle (Ct) is the cycle number at which thefluorescent signal of the reaction crosses the threshold.The Ct is used to calculate the initial DNA copy number,because the Ct value is inversely related to the startingamount of target. For example, in comparing real-timePCR results from samples containing different amounts oftarget, a sample with twice the starting amount will yield aCt one cycle earlier than a a sample with twice the numberof copies of the target, relative to a second sample, willhave a Ct one cycle earlier than that of the second sample.This assumes that the PCR is operating at 100% efficiency(i.e., the amount of product doubles perfectly during eachcycle) in both reactions.6
Basics of real-time PCRamplification product, which is present in a low amount,will not compete with the primers’ annealing capabilities.All of these factors contribute to more accurate data.SlopeThe slope of the log-linear phase of the amplificationreaction is a measure of reaction efficiency. To obtainaccurate and reproducible results, reactions should havean efficiency as close to 100% as possible, equivalent to aslope of –3.32 (see Efficiency, below, for more detail).Figure 4. Example of a standard curve of real-time PCR data. Astandard curve shows threshold cycle (Ct) on the y-axis and the startingquantity of RNA or DNA target on the x-axis. Slope, y-intercept, andcorrelation coefficient values are used to provide information about theperformance of the reaction.(y-axis). From this standard curve, information about theperformance of the reaction as well as various reactionparameters (including slope, y-intercept, and correlationcoefficient) can be derived. The concentrations chosenfor the standard curve should encompass the expectedconcentration range of the target in the experimentalsamples.Correlation coefficient (R2)The correlation coefficient is a measure of how well thedata fit the standard curve. The R2 value reflects thelinearity of the standard curve. Ideally, R2 1, although0.999 is generally the maximum value.Y-interceptThe y-intercept corresponds to the theoretical limit ofdetection of the reaction, or the Ct value expected if thelowest copy number of target molecules denoted on thex-axis gave rise to statistically significant amplification.Though PCR is theoretically capable of detecting a singlecopy of a target, a copy number of 2–10 is commonlyspecified as the lowest target level that can be reliablyquantified in real-time PCR applications. This limits theusefulness of the y-intercept value as a direct measure ofsensitivity. However, the y-intercept value may be useful forcomparing different amplification systems and targets.Exponential phaseIt is important to quantify your real-time PCR reaction inthe early part of the exponential phase as opposed to in thelater cycles or when the reaction reaches the plateau. At thebeginning of the exponential phase, all reagents are still inexcess, the DNA polymerase is still highly efficient, and theEfficiencyA PCR efficiency of 100% corresponds to a slope of –3.32,as determined by the following equation:1Efficiency 10(–1/slope) –1Ideally, the efficiency (E) of a PCR reaction should be 100%,meaning the template doubles after each thermal cycleduring exponential amplification. The actual efficiency cangive valuable information about the reaction. Experimentalfactors such as the length, secondary structure, and GCcontent of the amplicon can influence efficiency. Otherconditions that may influence efficiency are the dynamicsof the reaction itself, the use of non-optimal reagentconcentrations, and enzyme quality, which can result inefficiencies below 90%. The presence of PCR inhibitorsin one or more of the reagents can produce efficienciesof greater than 110%. A good reaction should have anefficiency between 90% and 110%, which corresponds to aslope of between –3.58 and –3.10.Dynamic rangeThis is the range over which an increase in startingmaterial concentration results in a corresponding increasein amplification product. Ideally, the dynamic range forreal-time PCR should be 7–8 orders of magnitude forplasmid DNA and at least a 3–4 log range for cDNA orgenomic DNA.Absolute quantificationAbsolute quantification describes a real-time PCRexperiment in which samples of known quantity are seriallydiluted and then amplified to generate a standard curve.Unknown samples are then quantified by comparison withthis curve.Relative quantificationRelative quantification describes a real-time PCRexperiment in which the expression of a gene of interestin one sample (i.e., treated) is compared to expressionof the same gene in a
asics of real-time PCR 1 1.1 Introduction 2 1.2 Overview of real-time PCR 3 1.3 Overview of real-time PCR components 4 1.4 Real-time PCR analysis technology 6 1.5 Real-time PCR fluorescence detection systems 10 1.6 Melting curve analysis 14 1.7 Passive reference dyes 15 1.8 Contamination prevention 16 1.9 Multiplex real-time PCR 16 1.10 Internal controls and reference genes 18
Applied Biosystems 7300/7500/7500 Fast Real‐Time PCR System Applied Biosystems 7900HT/7900HT Fast Real‐Time PCR System Applied Biosystems ViiA 7 Real‐Time PCR System QuantStudio 6 Flex Real‐Time PCR System QuantStudio 7 Flex Real‐Time PCR System QuantStudio 12k Flex System
The InsTAclone PCR Cloning Kit is compatible with all PCR buffers supplied by Thermo Scientific. Gel-analyze the PCR product for specificity and yield before cloning. Specific PCR products of 1 kb appearing as one discrete band on the gel can be used for ligation directly from PCR reaction mixture without any purification.
2 Brilliant III Ultra-Fast QRT-PCR Master Mix INTRODUCTION Quantitative reverse transcription PCR (QRT-PCR) is a powerful tool for gene expression analysis. The Brilliant III Ultra-Fast QRT-PCR Master Mix was developed for the ABI StepOnePlus and Bio-Rad CFX96 real-time PCR instruments and other fast-cycling systems (such as the ABI 7900HT and
SYBR Green PCR Master Mix and SYBR Green RT-PCR Reagents Kit User Guide 9 1 Product Information Purpose of the Kit The SYBR Green PCR Master Mix is a convenient premix of the components (except primers, template and water) necessary to perform real-time PCR using SYBR Green I Dye. Direct detection
Power SYBR Green PCR Master Mix and Power SYBR Green RT-PCR Reagents Kit User Guide 9 1 Product Information Purpose of the Kit The Power SYBR Green PCR Master Mix is a convenient premix of the components (except primers, template, and water) necessary to perform real-time PCR using SYBR Green I dye with enhanced sensitivity and specificity. The SYBR Green dye
PCR-HS-01W 96 0.1mL, Light Cycler Type, Low Profile White 10 100 Full Skirt PCR-FS-02960.2mL Clear10100 PCR-384-ROC 384 Roche Type,Two Notch Design, A24/P24 Clear 10 100 PCR-384-ROCW 384 Roche Type,Two Notch Design, A24/P24 White 10 100 PCR-384-ABI 384 ABI Type, Single Notch Design, A24 Clear 10 100
PrimePCR Assays, Panels, and Controls for Real-Time PCR 11 1 Introduction In 2012, Bio-Rad’s PrimePCR assays set a new standard for real-time PCR gene expression analysis. We collaborated with leaders in real-time PCR to design, optimize, and experimentally validate every primer pair. Products are available as individual SYBR Green
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