Good Practice Guide For The Application Of Quantitative .
Good practice guidefor the application ofquantitative PCR (qPCR)
Good practice guide forthe application ofquantitative PCR (qPCR)First Edition 2013Co-authorsTania Nolan (1)Jim Huggett (2)Elena Sanchez (2)ContributorsAnders Bergkvist (1)Malcolm Burns (2)Rebecca Sanders (2)Nicholas Redshaw (2)Tim Wilkes (2)AcknowledgementsWith special thanks to Susan Pang(2) andVicki Barwick(2) for their help in theproduction of this guide. Acknowledgementof an individual does not indicate theiragreement with this Guide in its entirety.Production of this Guide was in part fundedby the UK National Measurement System.Foreword by Michael Pfaffl(1)(2)(3)(3)Sigma-AldrichLGC, TeddingtonTechnical University Of MunichThis publication should be cited as:T Nolan, J Huggett, E Sanchez, Good practice guide forthe application of quantitative PCR (qPCR), LGC (2013).Copyright 2013 LGCi
Foreword by Michael W PfafflThe polymerase chain reaction (PCR) is a rapid, sensitive, and rather simple technique to amplifyDNA, using oligonucleotide primers, dNTPs and a heat stable Taq polymerase. It was invented in1983 by Kary B. Mullis and co-workers, who, ten years later, were awarded the ‘Nobel Prize forChemistry’. With the introduction of real-time PCR in the late nineties, the PCR method overcamean important hurdle towards becoming ‘fully quantitative’ (and therefore known as quantitativePCR, or qPCR). Currently, qPCR is regarded as the ‘gold standard’ in the quantitative analysis ofnucleic acids, be it DNA, RNA or micro-RNA molecules. The main reasons for its success are itshigh sensitivity, robustness, good reproducibility, broad dynamic quantification range, and veryimportantly, affordability. The assay and primer design can often be fully automated and handlingin the lab is blindingly easy.Another big draw for the user is that, in most instances, the qPCR experiments produce results,or as we call them, Cq data points. However, the generation of Cq data points is not dependenton good laboratory practice or the precise application of guidelines such as MIQE. In other words,when researchers obtain a Cq data point, they need to prove that that particular amplificationresult is valid, reliable and meaningful.And exactly here lies the main challenge of qPCR! This method is ‘too easy’ to apply andgenerates results any time. It is up to the researcher to demonstrate that the data obtained arevalid and if not, investigate where the error could come from.Hence, it is essential to have a comprehensive understanding of the underlying basic qPCRprinciples, sources of error, and general issues inherent to nucleic acid isolation and/orquantification in order to develop assays and workflows which meet high analytical requirementsin concordance with the MIQE guidelines. Unfortunately, we are still far from having developedsuch optimal workflows, with the highest sensitivity or the best RNA integrity metrics, to obtainreproducible and authentic results.Thus, I can warmly recommend to the research community this Good Practice Guide for theApplication of Quantitative PCR, with the aim to improve researchers’ experimental workflows,from sampling to qPCR data analysis, and eventually take us to valid and confident researchresults.Michael W PfafflMichael W. PfafflPhysiology WeihenstephanTechnische Universität MünchenWeihenstephaner Berg 385354 FreisingGermanyMichael.Pfaffl@wzw.tum.deii
Good Practice Guide for the Application of QuantitativePCR (qPCR)ContentsObjectives of this guide . 2Terminology . 3Common abbreviations . 7Section A. Technical information . 8A.1PCR and qPCR . 8A.2Sample purification and Quality Control (QC) . 10A.3qPCR assay design . 21A.4Assay optimisation and validation . 32A.5Normalisation . 41A.6Data analysis . 44A.7Troubleshooting . 50Section B. Applications of qPCR . 53B.1Use of PCR and qPCR in food authenticity studies . 53B.2Pathogen detection for clinical analysis . 59B.3MicroRNA expression profiling . 61Section C: Protocols . 64C.1Basic qPCR protocol . 65C.2The SPUD assay for detection of assay inhibitors . 68C.33’/5’ Assay for analysis of template integrity . 70C.4Reverse transcription (one-step and two-step) protocols . 72C.5Primer optimisation . 78C.6qPCR reference gene selection protocol . 84C.7qPCR efficiency determination protocol . 87C.8qPCR gene expression analysis protocols. 90Section D: Further resources . 94Section E: Further reading . 95Section F: References . 961
Objectives of this guideThere is little doubt that PCR (Polymerase Chain Reaction) has transformed the fields of clinicaland biological research, due to its robustness and simplicity. Subsequent developments, such asreal-time quantitative PCR (qPCR) and reverse transcription qPCR (RT-qPCR), offer simplemethods for analysis of DNA and RNA molecules. However, completing qPCR assays to a highstandard of analytical quality can be challenging for a number of reasons, which are discussed indetail in this guide.qPCR has a large number of applications in a wide range of areas, including healthcare and foodsafety. It is therefore of paramount importance that the results obtained are reliable in themselvesand comparable across different laboratories.This guide is aimed at individuals who are starting to use qPCR and realise that, while thismethod is easy to perform in the laboratory, numerous factors must be considered to ensure thatthe method will be applied correctly.These additional considerations include – but are not limited to – methods of sampling, samplestorage, nucleic acid extraction, and nucleic acid storage, manipulation and preparation. In otherwords, all the steps prior to undertaking the quantification technique must also be controlled. Atthe other end of the analytical process, reporting technical results may be highly subjective. SinceqPCR is a relative method, requiring the comparison of two or more samples to a standard curveor to each other, standardisation of results is very challenging. The primary qPCR metric, thequantification cycle (Cq)1, depends on many factors including where a threshold is set, the choiceof reporter and day-to-day variation in measurement. In addition, since Cq exists on a logarithmicscale, there are specific statistical challenges that need to be addressed to analyse these dataaccurately.All these factors combine to make a technically simple technique, challenging to interpret withabsolute confidence. This guide aims to assist those who are, or will be, using qPCR bydiscussing the issues that need consideration during experimental design. The guide entails “triedand tested” approaches, and troubleshoots common issues.2
TerminologyAbsolute (or Standard Curve) QuantificationAbsolute quantification is used when performing qPCR to describe estimation of target copynumbers by reference to a standard curve of defined, absolute concentration. This guide uses theterm standard curve quantification to describe this process.Allelic Discrimination (AD)Allelic discrimination assays are designed to define and differentiate genetic variants includingsingle nucleotide polymorphisms (SNPs). They use differentially labelled probes (one specific forthe wild type and another targeted for the mutant sequence), or a single probe to detect eitherproduct followed by a melt curve analysis to distinguish between the two. An alternative approachis to use DNA binding dyes in combination with melt curve analysis (see High Resolution Melt(HRM) analysis).AmpliconThe amplicon refers to the specific PCR product resulting from amplification of the primertargeted region.Amplification PlotThe amplification plot is a graph presenting the relationship between cycle number (x-axis) andfluorescence signal (y-axis). This results in a sigmoidal curve. Amplification, represented by theinitial log phase, is followed by a linear phase, and finally a plateau.CalibratorThe calibrator is a reference sample used as the basis for quantification studies.Comparative QuantificationComparative quantification is used to measure the relative change in expression levels betweensamples under different experimental conditions or over a period of time. The concentration of thegene target in each sample is compared to a validated reference gene or multiple referencegenes, to normalise for variations in sample loading. Comparative quantification is also known asrelative quantification.Cq (quantification cycle)1A generic term which includes Ct, (see below), crossing point (Cp), and all other instrumentspecific terms refering to the cycle used to quantify the concentration of target in the qPCR assay.Ct (threshold cycle)The Ct is defined as the number of cycles required to produce a constant emission offluorescence. The constant fluorescent emission is recorded relative to a defined thresholdsetting and the cycle number at which the fluorescence generated crosses the threshold is thereaction Ct. (N.B. The MIQE guidelines1 propose that Ct be replaced by Cq.)End Point AnalysisAn end point analysis is used to measure the amount of amplified product at the end of the PCR.Results are considered to be qualitative and are used to indicate the presence or absence of aspecific target sequence in a sample without the determination of target concentration.3
Endogenous Control/Reference Gene/NormaliserAn endogenous control is typically a gene target (or several in combination) present in eachsample at a constant concentration that is resistant to response fluctuations due to changes inbiological conditions. These have, historically been referred to as housekeeping genes. However,when measuring RNA, many targets can be used which cannot be considered as housekeepinggenes; hence the term of preference is now reference gene.Exogenous ControlAn exogenous (or external) control is a target sequence that is spiked into the sample at variousstages throughout the measurement process. Measuring the concentration of this spikedsequence can be used as a serial quality control and is widely used to monitor recovery efficiencyat different experimental stages, and to identify false negative results.Gene of Interest (GOI)The gene of interest is the gene target under investigation.High Resolution Melt (HRM)HRM is an extension of the traditional DNA melting analysis (see Melt Curve) and is used tocharacterise nucleic acid samples based on their dissociation (melting) behaviour. Samples canbe defined according to their sequence, length, G:C content or strand complementarity. Evensingle base changes such as SNPs can be identified. The technique requires a combination ofhigh-intensity optical detection systems, accurate thermal uniformity, high-speed data capture,extreme thermal resolution, DNA binding dyes, and dedicated software analysis.Melt CurveThe melt curve is a post-PCR analysis performed to estimate the specificity of amplified productsbased on their melting characteristics. Reactions performed in the presence of double-strandedDNA binding dyes are incubated through a range of increasing temperatures. When the meltingtemperature (Tm) of the amplicon is reached, the amplicon dissociates from a double-stranded toa single-stranded state leading to a drop in fluorescence of the double-stranded binding dye. Meltcurves are presented as a derivative plot showing the rate of change in fluorescent signal. The xaxis represents temperature and the y-axis displays the negative derivative (rate of change) offluorescence (F) with respect to temperature (T), shown as dF/dT. The melting temperature isdependent upon the length of the DNA sequence, G:C content and buffer. The peak of the plotrepresents the melting temperature (Tm) at which 50% of the DNA is single-stranded.MultiplexingMultiplexing is the process of simultaneous amplification and detection of more than one target ina single reaction tube. These reactions are usually detected using gene specific probes withdifferent fluorescent labels associated with each gene target. HRM can also be used to detectmultiple amplification products.No Template Control (NTC)No Template Control (NTC) qPCRs include all PCR reagents with the exception of the template.This is a standard negative control used to identify set-up contamination and primer-dimerproduct amplification.Normaliser or Reference GeneSee Endogenous Control.4
One-step Reverse Transcription (RT)-qPCRPrior to quantification of a target transcript, it is necessary to convert RNA to cDNA. Oneapproach is to perform both the reverse transcription and PCR amplification steps sequentially, inthe same tube. In a one-step RT-qPCR, gene-specific primers are used for both the RT and PCRsteps.Passive Reference DyeThe passive reference dye is added to the reaction mix to generate a signal for correction ofdifferences in optical sensitivity between distinct sample tubes. It also provides confirmation thatan equal volume of reaction mix has been added to each PCR sample. After baseline correction,the relative concentration of reference dye to signal intensity is traditionally plotted as dRn. Theabsolute intensity of the reference signal may influence data analysis since it directly affects thesignal to noise ratio.Quantification Cycle (Cq)1See CqQuencherThe quencher molecule is positioned in close proximity to the fluorescent label on a dual labelledprobe and absorbs the emission of reporter fluorescence, thus sequestering the signal output.Reaction Efficiency (E) or Amplification Value (A)The calculated rate of amplification is reported as a percentage, a fraction of 1 (for E) or fractionof 2 (for A). Efficiency calculations assume amplicon doubling during every cycle (for 100%efficiency). Efficiency (E) can be calculated using a standard curve with gradient m, using thefollowing equation (1)2:E 10(-1/m) –1(1)Reverse TranscriptionReverse transcription (RT) is the process of converting RNA to cDNA, using a reversetranscription enzyme and dNTPs.Reverse Transcription (RT) minus control (RT(-))The RT(-) control is used to identify contaminating sequences commonly derived from gDNAwithin a cDNA sample. The RT(-) control sample comprises all the components of the reversetranscription reaction, including target RNA, but with the omission of the reverse transcriptaseenzyme. This is another type of negative control (see NTC).Single Nucleotide Polymorphism – SNPSingle nucleotide polymorphisms (SNPs) are DNA sequence variants, or mutations, at a singlebase locus.Standard CurveA sample (also known as a calibrator) of known concentration units (e.g. pg/ L, copies/reactions,dilution factor, number of cells, or a relative dilution factor) is serially diluted through a controlledseries and used to construct a standard curve. The observations or measurements, in this caseCq values of these standards, are plotted against the logarithm of their concentration. Thestandard curve is used to predict analyte concentration of the unknown test samples from theobserved Cq.(see Absolute Quantification).5
Threshold Cycle (Ct) ValueSee Ct and Cq.Two-step RT-qPCRA two-step RT-qPCR is performed as two independent reactions: a reverse transcription reactionfollowed by qPCR. The reverse transcription step, using a blend of oligo-dT primers and randomoligonucleotides, produces a global (non-specific) cDNA population from all transcripts in theRNA sample. The cDNA is then used for subsequent analysis in a qPCR step and interrogatedfor the sequences of interest using gene-specific PCR primers.6
Common abbreviations(q)PCR: (quantitative) polymerase chain reactionA: AdenineC: CytosinecDNA: complementary DNAcfDNA: cell-free DNAdNTP: deoxyribonucleotide triphosphatedsDNA: double-stranded DNAG: GuaninegDNA: genomic DNAGOI: Gene of InterestmiRNA: microRNAmRNA: messenger RNAncRNA: non-coding RNARG: Reference GenerRNA: ribosomal RNART-qPCR: Reverse Transcription qPCRSDS: Sodium Dodecyl Sulfate (anionic detergent)ssDNA: single-stranded DNAT: ThyminetRNA: transfer RNAU: Uracyl7
Section A. Technical informationA.1 PCR and qPCRAn understanding of the polymerase chain reaction (PCR) is required to explain the technique ofreal-time quantitative polymerase chain reaction (qPCR). PCR is an immensely powerful, geneticamplification technique resulting in high levels of sensitivity and specificity in bioanalysis.PCR is an enzymatic reaction used to amplify DNA. DNA usually consists of a pair ofcomplementary polynucleotide strands linked together to form a double helix. The strands aremade up of the four bases: Adenine, Thymine, Guanine and Cytosine (abbreviated A, T, G, andC respectively) which pair with one other base, usually A to T, and C to G, on the complementarystrand. When a DNA solution is heated, the non-covalent, hydrogen bonds that hold the twostrands together weaken and eventually the two strands separate, denature or “melt”.PCR enables targeted regions of small amounts of DNA to be exponentially amplified, generatinglarger amounts of the target region. For example, 500 copies of a DNA sequence may beamplified to 50 billion copies, which is much easier to measure using currently availabletechnology. This reaction can be tailored in order to amplify specific sequences within the DNA,which are known as target sequences.TARGET DNAPrimer 2Primer 1Reaction containstarget DNA,primers and TaqpolymeraseThe reaction is heated to 95oC to denature target DNA. Subsequent cooling to aroun
Chemistry’. With the introduction of real-time PCR in the late nineties, the PCR method overcame an important hurdle towards becoming ‘fully quantitative’ (and therefore known as quantitative PCR, or qPCR). Currently, qPCR is regarded as the ‘gold standard’ in the quantitative analysis of
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