Gene quantification using real-time quantitative PCR(1).pdf

Experimental Hematology 30(2002)503512 0301-472X/02$see front matter.Copyright 2002 International Society for Experimental Hematology.Published by Elsevier Science Inc.PII S0301-472X(02)00806-8 Gene quantification using real-time quantitative PCR:An emerging technology hits the mainstream David G.Ginzinger Genome Analysis Core Facility,Comprehensive Cancer Center,School of Medicine,University of California at San Francisco,San Francisco,Calif.,USA(Received 10 December 2001;revised 20 February 2002;accepted 5 March 2002)The recent flood of reports using real-time Q-PCR testifies to the transformation of this tech-nology from an experimental tool into the scientific mainstream.Many of the applications ofreal-time Q-PCR include measuring mRNA expression levels,DNA copy number,transgenecopy number and expression analysis,allelic discrimination,and measuring viral titers.Therange of applications of real-time Q-PCR is immense and has been fueled in part by the prolif-eration of lower-cost instrumentation and reagents.Successful application of real-time Q-PCRis not trivial.However,this review will help guide the reader through the variables that canlimit the usefulness of this technology.Careful consideration of the assay design,templatepreparation,and analytical methods are essential for accurate gene quantification.2002 In-ternational Society for Experimental Hematology.Published by Elsevier Science Inc.A literature search with the keyword“quantitative PCR(Q-PCR)”or“real-time PCR”will generate literally thousandsof hits,testifying to the emergence of this technology intothe mainstream of many scientific disciplines.If the samesearch were to have been performed a few years ago,only acouple of hundred hits would have been returned.What hasaccounted for this sudden increase of papers utilizing thistechnology?The first documentation of real-time PCR wasin 1993 1,and yet this technology has only recently hit themainstream.Perhaps the primary reason for this was thegreat expense of the instruments,but it has certainly beenexacerbated by the complexities of performing reproduciblereal-time Q-PCR studies,and real-time PCR has only re-cently been widely accepted as a valuable technique.Withthe increasing number of real-time PCR thermocyclers onthe market and the decreasing prices of these instruments,aswell as the reagents,many more people now have access tothis technology.In order to design and analyze experimentsusing real-time PCR it is not sufficient to simply extendones knowledge of standard PCR,or even semiquantitativePCR.Many more controls are needed in order to be certainof results when using real-time PCR,as it differs signifi-cantly from simply looking for a band on a gel.The PCR reaction generates copies of a DNA template inan exponential fashion.Due to inhibitors of the polymerasereaction found with the template,reagent limitation,or ac-cumulation of pyrophosphate molecules,eventually thePCR reaction is no longer generating template at an expo-nential rate(otherwise known as the“plateau phase”),andsome reactions will generate more product than others.Thisis the most important reason that end-point quantitation ofPCR products is so unreliable.With the ability to measurethe PCR products as they are accumulating,or in“realtime,”it is possible to measure the amount of PCR productat a point in which the reaction is still in the exponentialrange.It is only during this exponential phase of the PCRreaction that it is possible to extrapolate back to determinethe starting amount of template.During the exponentialphase in real-time PCR experiments a fluorescence signalthreshold is determined at which point all samples can becompared.This threshold is calculated as a function of theamount of background fluorescence and is plotted at a pointin which the signal generated from a sample is significantlygreater than background fluorescence.Therefore,the frac-tional number of PCR cycles required to generate enoughfluorescent signal to reach this threshold is defined as thecycle threshold,or Ct.These Ct values are directly propor-tionate to the amount of starting template and are the basis Offprint requests to:David G.Ginzinger,Ph.D.,Genome Analysis CoreFacility,Comprehensive Cancer Center,School of Medicine,University ofCalifornia at San Francisco,2340 Sutter St.,Room S131,San Francisco,CA 94143-0808;E-mail:dginzingcc.ucsf.edu 504 D.G.Ginzinger/Experimental Hematology 30(2002)503512 for calculating mRNA expression levels or DNA copy num-ber measurements(Fig.1).What is real-time Q-PCR?Real-time quantitative PCR is the reliable detection andmeasurement of products generated during each cycle of thepolymerase chain reaction(PCR)process which are directlyproportionate to the amount of template prior to the start ofthe PCR process.To accomplish this it is necessary to havea method of detecting the accumulation of PCR product andan instrument in which to perform the thermocycling that isadapted to record the results during each PCR cycle in realtime.Early attempts to perform Q-PCR,prior to real-timeinstrument,relied on visualization of PCR products usingintercalation of ethidium bromide(or other intercalatingdyes)at an empirically determined PCR cycle number.These products were run on standard agarose gels and quan-tified with radio-imaging or other densitometric means.Theaddition of competitive PCR reactions provided some in-crease in the quantitative capacity,but together these meth-ods fell short of being convenient,robust,and reliable quan-titative assays.The first reported method of real-time PCR,by Higuchiin 1993 1,used ethidium bromide intercalation during the PCR process and a modified thermocycler to irradiate thesamples with ultraviolet(UV)light and then detected the re-sulting fluorescent signal with a charged coupled devise(CCD)camera.The fluorescent signal was plotted as a func-tion of cycle number.The resulting plot,now a very familiarsight(Fig.1),gave a good indication of the amount of PCRproduct that was generated during each cycle of PCR(exceptfor those early cycles that are beneath the detection limits ofthe CCD camera).The primary drawback to this approach,other than the use of a carcinogen like ethidium bromide,isthat nonspecific PCR products are equally detected and in-cluded in the total amount of fluorescent signal measured.Today,the chemistries most commonly employed in-clude 5?nuclease assays using TaqMan probes 2,3,molec-ular beacons 4,and SYBR Green I intercalating dyes 5,6(Fig.2).Other methods have also been reported 7,8;how-ever,in all cases a fluorescent signal is generated during thePCR process that is captured by any one of several differentreal-time instruments.Due to added specificity,the additionof a hybridization probe makes the real-time Q-PCR assaymuch more robust.The 5?nuclease assay generates a fluo-rescent signal by cleavage of a fluorescent molecule on the5?end of a target specific oligonucleotide(TaqMan probe,Applied Biosystems,Foster City,CA,USA).In native formthe 5?fluor is quenched by a second molecule on the 3?endof the probe.When excited by the light source,the intactprobe emits a signal in a wavelength characteristic of the 3?molecule due to Frster(or fluorescence)resonance energytransfer(FRET),which shifts the energy to be released aslight or as heat when using a black hole quencher(BHQ;Bio-search Technologies,Novato,CA,USA).A novel type of Taq-Man probe is available from Applied Biosystems which uses a“minor groove binding”(MGB)moiety on the 3?end thatraises the effective melting temperature(Tm)of the probe,thereby enabling the probe to be significantly shorter and en-hancing the function in an allelic discrimination assay 9.Moreover,these probes are capable of determining the expres-sion level of a specific allele to the exclusion of the opposite al-lele that differs by only one nucleotide(unpublished data).Currently there are numerous real-time thermocyclers onthe market;these include the ABI7700,ABI7900,andABI7000(Applied Biosystems),MX4000(Stratagene,LaJolla,CA,USA),Lightcycler(Roche,Alameda,CA,USA),iCycler(Bio-Rad,Hercules,CA,USA),Smartcycler(Cepheid,Sunnyvale,CA,USA),and the Robocycler(MJResearch,Incline Village,NV,USA).By far the most com-mon and the first to reach the mass market was the AB7700,which has recently been replaced with the ABI7000 orABI7900.One of the major advantages of the ABI instru-ments is the collection of data from a“passive reference”signal to normalize each reaction for variances in the opticsof the system.The MX4000 can also compensate for thisvariance,has software that is easy to use,and is able to per-form multiplex reactions,with up to three different PCR re-actions in a single tube.Although it is beyond the scope ofFigure 1.A hypothetical amplification plot illustrating the nomenclaturetypically used in real-time Q-PCR experiments.The amplification plot isthe plot of fluorescence signal vs PCR cycle number.The baseline isdefined as the PCR cycles in which a signal is accumulating but is beneaththe limits of detection of the instrument.The signal measured during thesePCR cycles is used to plot the threshold.The threshold is calculated as 10times the standard deviation of the average signal of the baseline fluores-cent signal.A fluorescent signal that is detected above the threshold is con-sidered a real signal that can be used to define the threshold cycle(Ct)for asample.The Ct is defined as the fractional PCR cycle number at which thefluorescent signal is greater than the minimal detection level.The Ct valuesof different samples are then used to calculate the relative abundance oftemplate for each sample.In this plot the solid line crosses the threshold atPCR cycle number 18 whereas the dotted line crosses at 20.By subtracting18 from 20,there is a two-cycle difference between these two samples or a?Ct of 2.Due to the exponential nature of PCR the?Ct is converted to alinear form by 2?(?Ct)or fourfold difference.This calculation is usedwhen performing a relative quantitation analytical method.D.G.Ginzinger/Experimental Hematology 30(2002)503512 505 this review article to compare and contrast all the instru-ments available on the market,one must be careful whenchoosing which instrument to buy.All of these instrumentsare capable of performing real-time PCR,yet they are notall equal.Cost should not be the only factor when making achoice;the cheaper models cannot compensate for the vari-ance in the optics and therefore are not capable of detectingsmaller differences.The higher-throughput instrument(e.g.,AB7900)may be more than you need;with a 384-well for-mat,it is capable of running a vast number of allelic dis-crimination assays.Great care and research should be givento your choice;it is imperative to match the instruments ca-pabilities with your needs.General applications of Q-PCR Applications of real-time Q-PCR are numerous.They in-clude:mRNA expression studies,DNA copy number mea-surements in genomic 1012 or viral DNAs 13,trans-gene copy number 14 and unpublished data,allelicdiscrimination assays 15,16 and confirmation of microar-ray data 1719.Some of the most recent applications,which demonstrate the sensitivity of this technology whenapplied to expression analysis of limited samples,includeexpression analysis of specific splice variants of genes 20and laser capture of microdissected material 21;paraffin-embedded tissues 22,23;flow-sorted cells,including stemcells(unpublished data);or randomly amplified RNA fromsingle cells 24.Specific applications ofreal-time Q-PCR to experimental hematology Real-time Q-PCR has been used in a small number of stud-ies recently published in this journal 2529.However,given the recent proliferation of studies in which real-timeQ-PCR has been used,it is only a matter of time before thefull potential of its application to hematology in realized.Specific examples include measurement of translocation gene but 6-FAM(6-carboxyfluorescein)is the most common one used.(B)Amolecular beacon.This is similar to a TaqMan probe in that it uses quenchingto prevent unwanted fluorescent signal,although through a direct transfer ofenergy.However,it differs in that it is not cleaved by the 5?exonucleaseactivity and only generates signal when hybridized to the template.The addi-tion of a hairpin is needed to provide the quenching effect.When hybridized,the distance of the 5?dye molecule from the quenching molecule(usuallyDABCYL)is great enough that there is very minimal quenching of the signal.Data must be analyzed during the annealing phase of the PCR.(C)SYBRGreen I DNA-binding dye.When using this method there is no need to designa third,modified oligonucleotide or hybridization probe.A fluorescent signalis only possible when the sample is excited by the light source when the dye isbound to the DNA molecule(possibly by intercalation in the minor groove ofdouble-stranded DNA).This is the cheapest of the methods but suffers fromthe need to optimize the PCR reactions such that the primers do not formprimer-dimers.SYBR Green I dye cannot discriminate between real templateand artifact bands,unlike that of TaqMan probes or molecular beacons.Figure 2.Methods used to generate a fluorescent signal in real-time Q-PCRexperiments.Although other methods exist 8,these are the most commonlyused in real-time Q-PCR studies.(A)A typical TaqMan probe used in a 5?nuclease assay to generate a signal using the 5?to 3?exonuclease activity ofTaq DNA polymerase.This probe is designed to hybridize to the templatebetween the standard PCR primers.Once cleaved from the rest of the probe,the 5?dye molecule is freed from the quenching effect(via FRET)when inclose proximity to a quenching molecule such as TAMRA(6-carboxy tetra-methyl rhodamine).The 5?dye can be one of several different molecules 506 D.G.Ginzinger/Experimental Hematology 30(2002)503512 products,DNA copy number measurements in leukemia,and analysis of mRNA expression levels of genes importantfor the differentiation of cell types of an immune response.Detection of minimal residual disease Hematological malignancies characterized by specific ge-netic translocations can be used as tumor markers whilemonitoring the response to therapy.Treatment options ofteninvolve chemotherapy,radiotherapy,and/or bone marrowtransplants.Over recent decades these advances havegreatly improved the chances for survival for these patients.However,the risk of recurrence remains a significant obsta-cle for complete remission.Thus,the detection of minimalresidual disease is a crucial step towards further refiningtreatment regimens 29,30.The use of real-time Q-PCR isbecoming a necessary research tool for detecting the molec-ular events underlying these recurrences and may guidetherapeutic decisions based on how individual patients re-spond at the molecular level.Thus quantitative measure-ments can be used to define correlations between theamount of fusion products and clinical outcome.In a specific example,the reciprocal translocationt