RT-PCRcanbeperformedaseitheratwo-steporanone-stepprocedure.Eachhascertainadvantages:

1.Two-stepprocedures

A.Twotube,two-stepprocedure.

Inthefirsttube,first-strandCDNAsynthesisisperformedunderoptimalconditions,usingeitherrandomhexamers,oligo(dT)primers(generatingacDNApool),orsequence-specificprimers.AnaliquotoftheRTreactionisthentransferredtoanothertube(containingThermostableDNApolymerase,DNApolymerasebuffer,andPCRprimers)forPCR.

Advantagesofthisapproach:ThismethodisusefulforexperimentswheremultipletranscriptshavetobeanalyzedfromthesameRTreactionorforspecificapplicationssuchasDifferentialDisplayReverseTranscription(DDRT)orRapidAmplificationofcDNAEnds(RACE).Also,sincetheRTreactionisperformedunderoptimalconditions,thisapproachproducesthelongestRT-PCRproducts(upto14kbinlength,iftheappropriateenzymesareused).

B.Onetube,two-stepprocedure.

Inthefirststep,reversetranscriptaseproducesfirst-strandcDNAinthepresenceofMg²⁺ions,highconcentrationsofdNTPs,andeitherspecificornon-specific[oligo(dT)]primers(reactionvolume,20µl).FollowingtheRTreaction,anoptimizedPCRbuffer(withoutMg²⁺ions),athermostableDNApolymerase,andspecificprimersareaddedtothetubeandPCRisperformed.Thisapproachmaybeusefulwhentemplateamountsarelimited,sincetheentireRTreactionisusedinthesubsequentPCR.

AdvantagesofTwo-stepRT-PCRProcedure.

Atwo-stepprocedurehasthefollowingadvantages:

• Optimizesreactionconditions.Thetwo-stepformatallowsbothreversetranscriptionandPCRtobeperformedunderoptimalconditionstoensureefficientandaccurateamplification.

• Providesflexibility.Two-stepproceduresallowtheproductofasinglecDNAsynthesisreactiontobeusedforanalysisofmultipletranscripts.ThisflexibilityisvaluableforsuchspecializedapplicationsasrapidamplificationofcDNAends(RACE)andDifferentialDisplayReverseTranscription(DDRT).

• Amplifieslongsequences.WiththerightcombinationofreversetranscriptaseandthermostableDNApolymerase,two-stepRT-PCRcanamplifyRNAsequencesupto14kblong.

2.Onetube,one-stepprocedure(coupledRT-PCR)

BothcDNAsynthesisandPCRamplificationareperformedwiththesamebufferandsite-specificprimers,eliminatingtheneedtoopenthereactiontubebetweentheRTandPCRsteps.Inadditiontothehighersensitivityofthisapproach(asintheonetube,two-stepreactionabove),theone-stepapproachminimizesthechanceofcontamination,sincetheentirereactionisperformedwithminimalpipettingstepsandwithoutopeningthetube.Inaddition,thisapproachpermitsdirectanalysisofaspecifictranscript,sincetheprimersusedinbothstepsaresequence-specific.Finally,thethermoactivereversetranscriptaseusedinthisprocedureallowahighRTreactiontemperature(50°–72°C),whichreducesfalseprimingandincreasesthespecificityofthereactionbyeliminatingsecondarymRNAstructure.

AdvantagesofOne-stepRT-PCRProcedure.

Aone-stepprocedurehasthefollowingadvantages:

• Minimizestimerequired.Theone-stepreactionhasfewerpipettingstepsthanthetwo-stepreaction,significantlyreducingthetimeneededtoperformRT-PCRandeliminatingpipettingerrors.

• Reducestheriskofcontamination.Theentireone-stepreactiontakesplaceinasingletube,withnotransfersrequiredandnoneedtoopenthereactiontube(stepswherecontaminationofthePCRsamplecantakeplace).

• ImprovesthesensitivityandspecificityofcDNAsynthesis.Twocharacteristicsoftheone-stepreactionprovideincreasedyieldandefficiency:(1)thecDNAreactionisperformedatahightemperature(toeliminateproblemswithRNAsecondarystructure),and(2)theentirecDNAsampleisusedastemplateforPCR.