Quantitative real-time PCR a powerful ally in cancer research(1).pdf

Quantitative real-time PCR:a powerful ally in cancer researchSimone Mocellin1,Carlo R.Rossi1,Pierluigi Pilati1,Donato Nitti1and FrancescoM.Marincola21Surgery Branch,Department of Oncological and Surgical Sciences,University of Padova,via Giustiniani 2,35128 Padova,Italy2Immunogenetics Laboratory,Department of Transfusion Medicine,Clinical Center,National Institutes of Health,10 Center Drive,Bethesda,MD 20814,USAIn this era of the Human Genome Project,quantitationof gene expression in tumor or host cells is of para-mount importance for investigating the gene patternsresponsible for cancer development,progression andresponse or resistance to treatment.Quantitative real-time PCR(qrt-PCR)technology has recently reached alevel of sensitivity,accuracy and practical ease that sup-ports its use as a routine bioinstrumentation for genelevel measurement.Several applications have alreadybeen implemented in the field of cancer research,andothers are being validated,showing that this molecularbiology tool can provide both researchers and clinicianswith precious information concerning the behavior oftumors.Knowledgeofthebiochemicalprinciplesunderlying this biotechnology can be of great value tointerpret correctly qrt-PCR data.PCR-based techniques enable us to obtain genetic infor-mation through the specific amplification of nucleic acidsequences,starting with a very low number of targetcopies.These reactions are characterized by a logarithmicamplification of the target sequences;that is,increase ofPCR copies followed by a plateau phase showing a rapiddecrease to zero of copy number increment per cycle.Accordingly,theamountofspecificDNAproductattheendof the PCR run bears no correlation with the number oftarget copies present in the original specimen.However,manyapplicationsinmedicineorresearchrequirequantification of the number of specific targets in thespecimen both to study the reaction of the cell or cellpopulation to a stimulus and to compare the gene profile ofdifferentsamples.AlthoughPCRanalysisgivesnoinformation on the biologically active products of genes(i.e.proteins),functional genomics studies have demon-strated a tight correlation between the function of aprotein and the expression patterns of its gene 1.Thisprovides a compelling reason for a gene profile-basedformulation of scientific hypotheses.Thefundamentalimportanceofgeneexpressionquantification methods in basic research,pharmaco-genomics and molecular diagnostics continues to directefforts aimed at improving current methodologies as wellas the development of novel technologies.Not all are basedon target amplification:the invader assay is a develop-ment of the invasive signal amplification assay thatcombines two signal amplification reactions in series togenerate and amplify a fluorescent signal in the presenceof the correct target sequence 2.However,reversetranscription(RT)-PCR-based assays are currently themost common method for characterizing or confirminggene expression patterns and comparing gene levels indifferent sample populations.Serial analysis of geneexpression(SAGE)allowsforhigh-throughputgeneprofiling 3.However,this technique is cumbersome,time-consuming and requires multiple manipulations ofthe samples,increasing the risk of carry-over contami-nation.Furthermore,similar to Northern and Southernblot,it requires large amounts of input mRNA,making theanalysis of hypocellular specimens impossible.Among the most promising innovations applied toconventional RT-PCR protocols is the development ofquantitative RT-PCR such as competitive standardizedRT-PCR and quantitative real-time PCR(qrt-PCR).Thesetechnologies present two major advantages:(1)the use ofstandardized competitor templates or standard curves,which permits comparison between experiments,and(2)the use of internal standards,which addresses the issue ofvariation in template starting amounts and operator-loading errors.Competitive RT-PCR is a time-consumingsystem,which is limited to sets of primers available fromone supplier.Furthermore,it does not eliminate the errorsassociated with individuals performing the reactions.Conceptual simplicity,practical ease and high-through-put capacity 4 have made real-time fluorescence detec-tion assay the most widely used gene quantificationmethod 5.In the field of oncology,qrt-PCR is experien-cing a rapid diffusion among investigators because of itspotential applicability to several research areas.Qrt-PCRpermits a highly sensitive quantification of DNA andtranscriptional gene levels in a few hours,with minimalhandling of the samples.The recent flood of reports usingqrt-PCR in cancer research testifies the transformation ofthis technology from an experimental tool into thescientific mainstream.PrinciplesThe concept of real-time PCR consists of the detection ofPCR products as they accumulate 6.Current qrt-PCRCorresponding author:Simone Mocellin().ReviewTRENDS in Molecular MedicineVol.9 No.5 May 2003189http:/1471-4914/03/$-see front matter q 2003 Elsevier Science Ltd.All rights reserved.doi:10.1016/S1471-4914(03)00047-9systems are based on a set of probe and primers,whichaccounts for the high specificity of the technique.Thedevelopment of fluorogenic probes eliminated the need forpost-PCR processing proper of previous systems 7.Twomain techniques are now available,which exploit theextension 8 or annealing 9 phase,respectively,togenerate fluorescence emission(Figs 1,2,3).In bothcases,the fluorescence signal increases with each PCRamplification cycle.The PCR cycle number at whichfluorescence reaches a threshold value of ten times thestandard deviation of baseline fluorescence emission isused for quantitative measurement(Fig.4).This cyclenumber is called the threshold cycle(Ct)and it is inverselyproportional to the starting amount of target geneticmaterial(Fig.5).By using probes labeled with differentfluorochromes characterized by unique emission spectra,more genes can be analysed at the same time within agiven sample(multiplex qrt-PCR)10.Although qrt-PCR analysis is sometimes referred to asabsolute gene quantitation,this term can be misleading.In fact,no matter what the source or how carefully it ismeasured,there is no way to know exactly how manycopies of a known template truly exist in a given well of aknown sample 11.A more appropriate term for thismethodisstandardcurve-basedquantitation,asastandard curve(fivefold or tenfold serial dilution)ofcalculated amount of a given gene is used to quantify thegene abundance in a sample of interest.Because both the amount of genetic material added toeachreversetranscriptionreactiontube(basedonwavelength absorbance)and its quality(i.e.degradation)are not reliable parameters to measure the startingmaterial,the number of copies of an endogenous controlgene generallyreferredtoashousekeepinggene isalsoquantified.Foreachexperimentalsamplethevalueofboththe target and the housekeeping gene are extrapolatedfrom the respective standard curve equation(Fig.5).Thetarget value is then divided by the endogenous referencevalue to obtain a normalized target value independentfrom the amount of starting material.The assumptionmustbemade thatthe chosenreference genedoesnot varyin copy number or expression level under differentexperimental conditions.Only if this assumption holdstrue will multiple samples be completely comparable.Main issuesNormalization of resultsThe identification of a valid reference for data normal-ization is a crucial issue in qrt-PCR experimental design.Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)isone of the most popular housekeeping genes,although itFig.1.Principles of quantitative real-time PCR using fluorogenic probes:schemeof the extension-phase method with standard probe.In addition to forward andreverse primers,this system uses a probe,which is an oligonucleotide with both areporter fluorescent dye(R)and a quencher dye(Q)attached at its 50and 30end,respectively.During the extension phase,the quencher can only quench the repor-ter fluorescence when the two dyes are close to each other.This is only the casefor an intact probe.In fact,once amplification occurs,the probe is degraded by the5030exonuclease activity of the Thermophilus aquaticus(Taq)DNA polymeraseand the fluorescence will be detected by means of a laser integrated in thesequence detector.TRENDS in Molecular Medicine R(a)(b)3535RQQFluorescence emissionProbeTaq polymeraseTarget geneTarget geneFig.2.Principles of quantitative real-time PCR using fluorogenic probes:schemeof the extension-phase method with beacon probe.Molecular beacons are hairpin-shaped molecules with an internally quenched fluorophore whose fluorescence isrestored when they bind to a target nucleic acid.They are designed in such a waythat the loop portion of the molecule is a probe sequence complementary to a tar-get nucleic acid molecule.The stem is formed by the annealing of complementaryarm sequences on the ends of the probe sequence.A fluorescent moiety(R)isattached to the end of one arm and a quenching moiety(Q)is attached to the endof the other arm.The stem keeps these two moieties in close proximity to eachother,causing the fluorescence of the fluorophore to be quenched.When theprobe encounters a target molecule,the molecular beacon undergoes a spon-taneous conformational reorganization that forces the stem apart,and causes thefluorophore and the quencher to move away from each other,leading to the res-toration offluorescence.TRENDS in Molecular Medicine R3355QRQTarget geneTarget geneBeacon probeFluorescenceemission(a)(b)ReviewTRENDS in Molecular MedicineVol.9 No.5 May 2003190http:/has been documented that GAPDH mRNA levels are notalways constant 12,particularly under the same patho-logical conditions 13.We and other authors routinely useb-actin as housekeeping gene 14,15.Even though theissues regarding b-actin gene regulation and pseudogeneexistence have been raised 16,17,the consistency ofresults yielded over time supports ex adjuvantibus the useof this reference gene.Alternatively,rRNA,which makesup the bulk of a total RNA sample,is another normalizerthat has been proposed 16,despite reservations concern-ing its expression levels,transcription by a different RNApolymerase and possible imbalances in rRNA and mRNAfractions between different samples 18.Other investi-gators have advocated normalization to total cellular RNAas the least unreliable method 19.However,little isknown about the total RNA content per cell of differenttissues invivo,or how thismight vary betweenindividualsor between normal and tumor tissue.To minimize thepotential variability characteristic of each single house-keeping gene,some investigators have recently proposedthe normalization of qrt-PCR data by geometric averagingof a set of reference genes 20.mRNA cell sourceWhen dealing with cell lines or in vitro purified cellpopulations the issue of gene expression normalization isstrictly about the best way to measure correctly gene copynumber.Ex vivo samples present an additional problemregarding qrt-PCR data interpretation.In fact,untilrecently,in vivo RNA extractions and subsequent analysescould only be performed from whole-tissue biopsies withlittle regard for the different cell types contained withinthat sample.This inevitably results in the averaging of theexpression of different cell types and the expression profileof a specific cell type might be masked,lost or ascribed toand dismissed as illegitimate transcription because of thebulk of the surrounding cells.This is particularly relevantwhen comparing gene expression profiles between normaland cancer tissue because normal cells adjacent to a tumormight be phenotypically normal,but genotypically abnor-mal or exhibit altered gene expression profiles because oftheir proximity to the tumor 21,and some tumors havesignificantly larger immune cell infiltrates than others22.Recent technological developments might bring asolution to this important issue.In particular,theintroduction of laser capture microdissection representsa crucial step forward 23,24,permitting the extraction ofa pure subpopulation of cells from heterogeneous in vivocell samples for detailed molecular analysis 25.Further-more,after the introduction of RNA linear amplifica-tion26,theissueoflimitedamountofgeneticmaterial obtained from tissue microdissection can beeasily overcome.Because this RNA method is charac-terized by a 50-biased gene amplification,particularattention must be paid to probe or primer design,so thatthey span the 30-flank of a given transcript sequence.Fig.3.Principles of quantitative real-time PCR using fluorogenic probes:schemeof the annealing phase method.In this case,two different probes are used,onecarrying a fluorescent reporter at its 30end(R1),whereas the other carries anotherfluorescent dye at its 50end(R2).The sequences of these two oligonucleotides areselected such that they hybridize to the amplified DNA fragment in a head-to-tailarrangement.When the oligonucleotides hybridize in this orientation,the two flu-orescence dyes are positioned in close proximity to each other.The first dye(R1)is excited by the filtered light source and emits a fluorescent light at a slightlylonger wavelength.When the two dyes are in close proximity,the energy emittedby R1 excites R2 attached to the second hybridization probe,which subsequentlyemits fluorescent light at an even longer wavelength.This energy transfer isreferred to as fluorescence resonance energy transfer(FRET).Choosing the appro-priate detection channel,the intensity of the light emitted by R2 is filtered andmeasured.TRENDS in Molecular Medicine 3553R2R1Fluorescenceemission+EnergytransferLightexcitationR2FluorescenceemissionProbesannealingTarget geneTarget geneR2R1R1(a)(b)Fig.4.b-actin amplification plot illustrating the nomenclature typically used inquantitative real-time PCR experiments.The amplification plot is the plot of fluor-escence signal versus PCR cycle number.The signal measured during these PCRcycles is used to plot the threshold.The threshold is calculated as ten times thestandard deviation of the average signal of the baseline fluorescent signal.A fluor-escent signal that is detected above the threshold is considered a real signal thatcan be used to define the threshold cycle(Ct)for a sample.The Ct is defined as thefractional PCR cycle number at which the fluorescent signal is greater than theminimal detection level.The Ct values of different b-actin concentrations are usedto generate the standard curve and then calculate the relative equation(Fig.4).TRENDS in Molecular Medicine 0.010.11100510152025303540PCR cycle numberFluorescence intensity(log scale)PCR plateauTemplatecopiesFluorescencethreshold level108107106105104103ReviewTRENDS in Molecular MedicineVol.9 No.5 May 2003191http:/Applications in cancer researchTherangeofqrt-PCRapplicationsinthefieldofoncologyisimmense and has been fuelled in part by the proliferationof low-cost instrumentation and reagents.The followingparagraphs arenotmeanttobeacomprehen