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Competitive RTPCR Strategy for Quantitative Evaluation of...

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Quantizationofgeneexpressionrequiresthatanaccuratemeasurementofaspecifictranscriptismade.Inthispaper,aquantitativereversetranscription-polymerasechainreaction(RT-PCR)bycompetitionfortilapiagrowthhormonereceptortypeIisdesignedandvalidated.ThisexperimentalprocedurewasusedtodeterminetheabundanceofgrowthhormonereceptortypeItranscriptindifferenttilapiatissues.TheresultsobtainedwiththisdevelopedcompetitiveRT-PCRweresimilartoreal-timePCRresultsreportedrecently.Thisprotocolprovidesareliablealternative,butlessexpensivethanreal-timePCRtoquantifyspecificgenes.

KeyWords:Tilapia-Receptors-Somatostatin

Introduction

Growthhormone(GH)playsacentralroleasapluripotentendocrineregulatorofphysiologicalfunctionsinfishandhighervertebrates,workingthroughspecificcellmembranereceptor(GHR)thattriggersaphosphorylationcascadeforsignalingandgeneexpressionevents(1,2).ThenucleotidesequenceofGHRisavailableformammals,birds,reptiles,andXenopus.Basedonconservedstructuralfeatures,thesereceptorsbelongtoclassIcytokinereceptorsuperfamilythatinclude,amongothers,receptorsforprolactin,erythropoietin,granulocytecolonystimulatingfactor,andseveralinterleukins(3).Sincetheinitialcloningandsequenceofgoldfish(Carassiusauratus)(4)andturbot(Scophthalmusmaximus)(5)GHRs,otherfishGHRshavebeencharacterizedinblackseabream(Acanthopagrusschlegeli)(6),giltheadseabream(Sparusaurata)(7),masusalmon(Oncorhynchusmasou)(8),rainbowtrout(Oncorhynchusmykiss)(9),catfish(Silurusmeridionalis),andtilapia(Oreochromisniloticus)(10).Aminoacidalignmentoffull-lengthGHRsrevealsarelativehighdegreeofidentity(35–40%)amongtetrapodsandnon-salmonidfishGHRs(GHRtypeI).SeveralauthorshavepostulatedadivergentevolutionofsalmonidGHRs(GHRtypeII);however,ithasrecentlybeenclonedaGHRinrainbowtrout(Oncorhynchusmykiss),whichisanalogoustoGHRsofnon-salmonidfish(GHRtypeI)andaGHRtypeIIinnon-salmonidfish(11).DuplicatedfishGHRsrepresentanewandperhapscomplexstepontheregulationoffishsomatotropicaxis.Inthisscenario,accuratemeasurementsofbothGHRexpressionpatternsindifferenttissuesandindifferentphysiologicalstagesarenecessary.Theclassicmethodstodothis,suchasNorthernblotsandRNAseprotectionassay,havebeenimprovedovertheyearsandhaveprovidedreliableresults.However,theysharetheweaknessofhavingtoolowsensitivityamongotherdrawbacks.Becauseofitsextremesensitivity,thepolymerasechainreaction(PCR)hasthepotentialtodetectandpreciselyquantifyspecificRNAsequencesifitisusedincombinationwithreversetranscription.However,therepetitivemultiplicationoftemplatemoleculesisadrawbackforquantitativemeasurementsbecausesmalldifferencesinthemultiplicationfactorleadtolargedifferencesintheamountofproduct(12).AlthoughtheuseofPCRforquantificationhasbeenuncriticallyacceptedbymanyscientists,itreallycannotberelieduponforquantitativemeasurements.Twomethodscanbeusedtosolvetheproblemofquantification:kineticmethodsandco-amplificationmethods.Co-amplificationmethodscanbedonewithoutexpensiveequipment.Inthisstudy,wedesignacompetitormoleculetoquantifyaccuratelythetilapiagrowthhormonereceptortypeI(tiGHRI)indifferenttilapiatissuesusingaquantitativeRT-PCRbycompetitionandweshowthatitissensitive,reproducIBLe,androbust.

MaterialsandMethods

CloningaTiGHRIProbe

Wedesignedfourforwarddegenerateoligonucleotides(A,B,C,andD)thatcontainedallthecodingsequencesforaconservedNglycosilationsiteoftheGHRextracellulardomain(LNWTLLNI)andfourreversedegenerateoligonucleotides(E,F,G,andH)thatcontainedallthecodingsequencescorrespondingtotheproline-richsiteintheintracellulardomainboxI(PKIKGIDP)(Table1).

Table1ForwardandreversedegenerateoligonucleotidestoclonetiGHRIprobe

TotalRNAfromtilapialiver(O.niloticus)wasobtainedbytheacidphenolmethod(13).MessengerRNAwaspurifiedfromtotalRNAusingthe“PolyAtract®mRNAIsolationSystemIII”kit(Promega,USA).MessengerRNAwasreversetranscribedwitholigo(dT)15using“ReverseTranscriptionSystem”(Promega).Polymerasechainreactionsweresetupin50-μlvolumesusing“PCRMasterMix”(Promega)with3μMofforwardandreverseprimersand1/10volumeoftheRTreaction.Weusedallpossiblecombinationsofthedegeneratedoligonucleotides(16differentreactions).ThePCRconditionused95°Cfor3min,followedbyacyclingprogramof94°Cfor1min,42°Cfor1minand72°Cfor1minfor30cycles,andafinalextensionat72°Cfor5min.PCRproductswerepurifiedfromagarosegelusing“Qiaquick®GelExtraction”Kit(Qiagen,USA)andclonedinT-vector(pGEM®-TEasyVectorSystemI,Promega).Theselectedclonesweresequencedusingstandardtechniques(14).

GenerationofCompetitor

Startingwithaclonecontaininganinsertof458bpoftiGHRI(Probe),wedidtwosubcloningstepstoobtainaninternalduplicationof100bprespecttooriginalfragment,generatingafragmentusedascompetitor(Fig.1).ThecompetitorwaslinearizedwiththeendonucleaseSmaIandtranscribedinvitrousing“T7RiboMAXExpressRNAiSystem”kit(Promega)toobtainthecompetitorRNA.

MediaObjects/12575_2009_9002_Fig1_HTML.gif

Fig.1DesignfortheisolationofprobefromthetiGHRICDNAandconstructionofcompetitorfragmenttouseinquantitativePCR.aDiagramofprobeamplificationfromthetiGHRIcDNAusingdegenerateoligonucleotideBandE.ECD,extracellulardomain;TMD,transmembranedomain;ICD,intracellulardomain;blackbox1,conservedNglycosilationsiteoftheGHRextracellulardomain(LNWTLLNI);blackbox2,codingsequencescorrespondingtotheproline-richsiteintheintracellulardomainboxI(PKIKGIDP);Probe,458-bpDNAfragmentoftiGHRIobtainedwiththeB–EoligonucleotidemixesandclonedinEasyT-vector(shadowboxes);Competitor,473DNAfragmentobtainedfromtwosubcloningstepstoproduceatandemoftwocopiesofthePstI–AccIfragmentoftheProbe;IandJ,internaloligonucleotidefromtheprobefragmentdesignedfortheamplificationoftargetandcompetitor;E,EcoRI;P,PstI;A,AccI;S,SacI.b2%agarosegelimagewiththePCRamplificationproductsusingoligonucleotidesIandJ:1,PCRnegativecontrol(withouttemplate);2,PCRusingaRTreactionfromtilapialivermRNAastemplate;3,PCRusingcompetitorsequenceastemplate,4,PCRusingprobeastemplate.

CompetitivePCR

Wedesignedtwospecificoligonucleotides(I = ccccacctactgctgatgttagandJ = caggaacaggcggcagcagg)thathybridizeinsidetothefragmentofthetiGHRgeneclonedbetweenbindingsitesofdegenerateoligonucleotides.WhenweusethesespecificoligonucleotidesinaPCR,wegeneratea366bpamplificationproductfromthewild-typeDNA(T-target)anda473bpamplificationproductfromthecompetitorDNA(Ccompetitor)(Fig.1b).ThePCRreactionsweresetupin50µlwith“PCRMasterMix”and0.2µMofeachprimer.Weusedadenaturalizationstepof95°Cfor2min,followedbyacyclingprogramof94°Cfor30s,62°Cfor30s,and72°Cfor1minfor30cycles.APCRnegativecontrolwassetupforallthePCRbatchestoascertaintheauthenticityofPCR.Theamplificationproductswereresolvedin2%agarosegelswithethidiumbromide.GelimageswereobtainedusingadigitalcameraOlympusC7070WideZoom.Thephotossavedinjpegformatwereusedfordensitometryanalysis.

QuantitativeAnalysis

ThedensitometrydataforbandintensitiesindifferentsetsofexperimentswasgeneratedbyanalyzingthegelimagesontheImageJprogram(Version1.33,USA).PreviouslytotheexperimentsofcompetitivePCR,wedidanexperiment(datanotshown)tocontroltheconsistencyofourdensitometryrawdata.Becauseofthelowdynamicrangeofethidiumbromidegels,itisnecessarytocontrolifthepeakareascorrespondingtodensitometryvaluesobtainedbyImageJprogramreproducereallythebandintensities.Inthisexperiment,weusedawiderangeofconcentrationofDNAandconsideredtherelationdose–response.Thelinealrelationislostafter100ngofDNA.

DeterminationofTarget/competitorAmplificationEfficiency

TenidenticalPCRmixtureswereprepared,asdescribedabove,eachcontaining100,000moleculesoftargetandcompetitorDNA.ThePCRcyclingconditionswerecarriedthrough45cycleswithonetubebeingremovedafter17,19,21,24,27,30,33,36,39,and45cyclesandtheamountofthePCRproductsquantified.Procedurewasrepeatedtwotimes.Efficiencywascalculatedas $${ ext{Ei}} = {{left( {{ ext{Pi}} - { ext{Pi}} - 1}
ight)} mathord{left/ {vphantom {{left( {{ ext{Pi}} - { ext{Pi}} - 1}
ight)} {{ ext{Pi}} - 1}}}
ight. kern-
ulldelimiterspace} {{ ext{Pi}} - 1}}$$ (12),whereEiistheefficiencyinonestep,Piisthequantityofproductinthatstep,andPi − 1istheproductalreadyaccumulatedduringthepreviousstep.Theefficiencymeansfortargetandcompetitorineachcyclewerecomparedusingmatchedttest.

DeterminationofAccuracyoftheCompetitivePCR

TotesttheprecisionoftheresultsobtainedwiththiscompetitivePCR,fivedifferentamountsofT(10,000,100,000,150,000,200,000,and1,000,000molecules)wereassayedwithserialdilutionsoftheC.Eachsetofvalidationexperimentscomprisedatleastfourreactioncombinations(TrelatedtoC)withthreereplicasforeachpointintheconditionsdescribedabove.Oneoftheseexperiments(100,000moleculesoftargetwithsixcompetitordilutionswiththreereplicasofeachpoint)wasrepeatedthreetimesindifferentdays.Theseproducedadatasetof120reactionstoaddresstheintra-andinter-experimentvariABIlity,precision,andresolutionofourexperimentalsystem.

SensitivityofCompetitivePCR

TotesttheminimumquantityoftargetmoleculesthatourPCRisabletodetect,wedidfivesetsofexperimentsrangingfrom2,000until10moleculesoftargetwiththreedifferentdilutionsofcompetitorwithtworeplicasforeachpointintheconditionssetupabove.

CompetitiveRT-PCR

TodeterminethetiGHRIexpressionlevelsindifferenttilapiatissues,westartedfromtotalRNAmini-preparationsofeachtissueusingtheacidphenolmethod(13).Weusedthreejuveniletilapias(O.niloticus)of100gassourceof100mgoftissuefromliver,muscle,brain,heart,stomach,spleen,intestine,andgonads.TheRT-PCRreactionsweredoneusing“Ready-To-Go™RT-PCRBeads”(AmershamBiosciences,USA)usingthesamecyclingprofiledescribedbefore.ForeachsampleoftotalRNA,weusedatleasttwoknowndifferentquantitiesofcompetitorRNAmoleculestoobtainlinearregressions.Wedeterminedthenumberoftargetmoleculesinthesamplewhenlog(T/C)equalszero(15).Inthisway,thetargetmoleculesnumberforalltissuesamplesofeachtilapiawasobtained.ThesevalueswerenormalizedversustotalRNA(RNAt)usedtodotheRTreaction.ThesamequantityofRNAtusedintheRTreactionwaselectrophoresedon1.5%formaldehydeagarosegel.Thedensitometrydataofthebandscorrespondingtothe28SsubunitsmeasuredwithImageJprogramwereconvertedtomicrogramsofRNAtusingareferenceRNAtwithknownconcentration.Then,theresultswereexpressedasnumberoftiGHRImolecules/µgRNAt.TheobtainedaveragesofthetiGHRImolecules/µgRNAtforeachtissuewerecomparedbynon-parametricKruskal–WallistestfollowedbyDunnmultiplecomparisontest(Prism,version4.0forWindows;GraphPadSoftware,USA).

Results

CloningTiGHRIProbe,ConstructionofCompetitor,andVerificationofDifferentiableAmplification

Thecombinationbetweenthefourforwarddegeneratedoligonucleotides(A,B,C,andD)andthefourreversedegeneratedoligonucleotides(E,F,G,andH)give16differentPCRs.Thebandsnearby500bpwereclonedinT-vectorandsequenced.Oneclone(probe)witha458bpfragmentoftiGHRIwasobtainedwiththeB–Eoligonucleotidemixes(Fig.2).Thisfragmentis100%identicaltothereportedGHRtypeIfromtilapia(O.niloticus)(accessionnumberAY973232),withoutthesequenceofthedegeneratedoligonucleotides.Afterthecompetitorconstruction,wewereabletoseeadifferenceintheelectrophoreticmobilitybetweentheamplificationproductsofwild-typeandcompetitorsequencesoftiGHRIusingIandJoligonucleotidesinthePCR(Fig.1b).

MediaObjects/12575_2009_9002_Fig2_HTML.gif

Fig.2Sequenceoftheprobecorrespondingtoa458-bpfragmentoftiGHRI.Inboldandunderline,oligonucleotidescorrespondingtomixBandE,respectively;insidebox,aminoacidsequencesusedtodesigndegeneratedoligonucleotides.

VerificationofEqualAmplificationEfficienciesforTargetandCompetitor

TargetandcompetitoramplifyinthecompetitivePCRwiththesameefficiencyover24–39cycles(Fig.3).Nostatisticallysignificantdifferencesbetweenthecycleefficiencyaveragesofthecompetitorandtargetsequenceswereencounteredusingapairedttest(p < 0.05).AllsubsequentcompetitivePCRswerecarriedoutfor30cycles,whentheamplificationwasfoundtobelinealandtheefficiencywasdiminishingbutitwasequalforthetargetandcompetitorsequences.Inaddition,to30cyclesthequantityofamplificationproductsinourPCRconditionsissufficientlyvisibleinanethidiumbromidegelandkeepsthelinealrelationshiptomeasuredpeakareas.Itmeansthatthebandintensitiesarefarfromthegelsaturationpoint.

MediaObjects/12575_2009_9002_Fig3_HTML.gif

Fig.3PCRefficienciesoftargetandcompetitor.Inthepicture:1,PCRnegativecontrol;amplificationproductsat17,19,21,24,27,30,33,36,39,and45cyclescorrespondingtolines2,3,4,5,6,7,8,9,10,and11,respectively,using10⁵moleculesofcompetitorandtargetinthePCR;12,molecularweightMarker(fromtoptobottom = 1.4kb,1.2kb,750bp,450bp,and366bp).Linesrepresentdensitometrydataofthetargetandcompetitorbandintensitiesversuscyclenumber.Eachpointistheaveragebetweentwodifferentexperiments.BarsrepresentthetargetandcompetitorPCRefficienciesforeachcycle.Therearenostatisticallysignificantdifferencesbetweentargetandcompetitorefficienciesinanycyclesdeterminedusingmatchedttest(p > 0.05).

AccuracyofCompetitivePCR

Therawdatacollectionofthe120amplificationreactionsisgiveninfile1oftheElectronicsupplementarymaterial.Fivedifferentquantitiesofthetargetsequence(10,000,100,000,150,000,200,000,and1,000,000molecules)wereamplifiedinthepresenceofdilutionseriesofthecompetitorsequence.Thelogratioofthequantityofproductsversusthelogofthecompetitorsequenceaddedwasplotted(Fig.4).ThevalidityofthecompetitionreactiondataatdifferenttargetconcentrationsisrepresentedbyregressionequationswithrespectiveR²values.Eachpointinthebestfittedregressionlineistheaverageamongthreereplicas.Thecalculatedconcentrationofthetargetsequenceisequaltotheconcentrationofcompetitorsequence,determinedfromtheregressionline,whenthelogratiooftheproductsequalszero.ThereisagoodcorrelationbetweentheobservedandexpectedconcentrationsofthetargetsequencecharacterizedbyaPearsonrof0.9978(95%CI0.9656–0.9999)withp < 0.001extremelysignificant(Table2).Intra-andinter-experimentrepeatabilitywasmeasured.ThecoefficientsofvariationamongthereplicasoftheexperimentallydeterminedtargetconcentrationsineachexperimentareshowninTable3.Theexperimentusing100,000moleculesoftargetsequencewithsixdifferentdilutionsofthecompetitorsequencewiththreereplicasbyeachpointwasrepeatedthreetimesindifferentdays.Triplicateanalysesyieldedanexperimentallydeterminedquantityoftargetof107,000 + 8,622moleculeswithaninter-experimentcoefficientofvariationof8%.

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Fig.4LinealregressionsgeneratedfromthedensitometrydataofthePCRreactionusingfixedtargetmoleculenumbersandserialdilutionsofcompetitor.Inthepicture,typical2%gelimageofamplificationproductsinthecompetitivePCRswhere10⁵moleculesoftargetwereusedwithserialdilutionsofcompetitor.1,PCRnegativecontrol;lanes2–4,10⁴;lanes5–7,5 × 10⁴;lanes8–10,7.5 × 10⁴;lanes11–13,10⁵;lanes14–16,5 × 10⁵;lanes17–19,7.5 × 10⁵moleculesofcompetitor,respectively;20,molecularweightmarker(fromtoptobottom = 1.4kb,1.2kb,750bp,450bp,and366bp).

Table2Numberoftheexpectedandobservedtargetmolecules

Oligonucleotides	Sequences
A	5′…(tc)t(ag)aa(ct)tggac(acgt)(tc)t(ag)tt(ag)aa(ct)at…3′
B	5′…ct(tc)aa(ct)tggac(acgt)tt(ag)(ct)t(ag)aa(ct)at…3′
C	5′…(ct)t(ag)aa(ct)tggac(acgt)ct(tc)ct(tcag)aa(tc)at…3′
D	5′…ct(tc)aa(ct)tggac(acgt)ct(tcag)ct(tc)aa(tc)at…3′
E	5′…gg(ga)tc(tag)at(tg)cc(tc)tt(tga)at(tc)tt(tg)gg…3′
F	5′…gg(ga)tc(tga)at(tg)cc(tc)tt(tga)at(tc)tt(ca)gg…3′
G	5′…gg(ga)tc(tag)at(ca)cc(tc)tt(tga)at(tc)tt(tg)gg…3′
H	5′…gg(ag)tc(tag)at(ca)cc(tc)tt(tga)at(tc)tt(ca)gg…3′

^aCharacterizedbyaPearsonrof0.9978(95%CI0.9656–0.9999)withp < 0.001

Table3Intra-experimentvariability

Numberoftargetmolecules^a
Expected	Observed
10,000	11,000
100,000	107,000
150,000	146,000
200,000	290,000
1,000,000	1,180,000

^aExperimentsA,B,andCcorrespondtoexperimentsdevelopedonthreedifferentdays

^bEachvaluecorrespondstothemeanofthreeexperimentalreplicates

SensitivityofCompetitivePCR

Sevenhundredandfiftymoleculesin50µlofPCRisthelowerlimitofquantification(LLQ)oftargetDNAsequencethatthecompetitivePCRwasabletodetect.

CompetitiveRT-PCRinDifferentTilapiaTissues

AbundancelevelsoftiGHRImRNA(target)indifferenttilapiatissuesweremeasuredusingthequantitativeRT-PCRbycompetitionvalidatedabove.LiverRNAofthreedifferenttilapiaswasassayedinthepresenceofdifferentquantitiesofcompetitorRNA.DensitometrydataderivedfrombandintensitiesofcompetitorandtargetforeachtilapiawereplottedasdescribedinSection2(Fig.5).WediscardedtheamplificationofgenomicDNAsequencesbecausethespecificoligonucleotidesIandJhybridizetodifferentexons,whichmeansthat,inthegenomicDNAsequencesbetweentheseoligonucleotides,thereareintrons.PCRamplificationsfromgenomicDNAsequenceswouldgiveahighersizethan366bpand473bpexpectedfortargetandcompetitoramplifications,respectively.ThequantityofspecificRNAobtainedforeachtilapialiverwasnormalizedversusthebandintensityofthe28SsubunitofthetotalRNAusedintheRTreaction.ThequantityoftiGHRIRNAreportedhereforliveristheaverageofthenormalizeddataobtainedfromeachtilapia.ThecoefficientofvariationispresentedintheElectronicsupplementarymaterial2.Theprocedurewiththeothertilapiatissueswasthesame.Therawdatacanbeseentooinfile2oftheElectronicsupplementarymaterial.Thecoefficientsofvariationoftheseexperimentswerebetween10%and50%.Aftertheprocessingofallobtaineddatafromtheselectedtilapiatissues,weobtainedtheprofileoftheexpressionlevelsoftiGHRImRNA(Fig.6).

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Fig.5DeterminationofthequantityoftiGHRIRNAintilapialiver.Linealregressionswithdatafromthreedifferenttilapias.Thepictureisanexampleof2%agarosegelwiththeamplificationproductsofcompetitiveRT-PCRusingtotalRNAfromtheliveroftilapia4andknownquantitiesofcompetitorRNA.1,molecularweightmarker(leader100bp,Promega);2,RT-PCRnegativecontrol;3,RT-PCRwith5 × 10⁵moleculesofRNAcompetitoralone;4,RT-PCRwithlivertotalRNAoftilapiaalone;5,6,and7,RT-PCRoflivertotalRNAoftilapia4with3 × 10⁵,6 × 10⁵,and8 × 10⁵moleculesofcompetitor,respectively.

MediaObjects/12575_2009_9002_Fig6_HTML.gif

Fig.6ExpressionoftiGHRIindifferenttilapiatissues.Thenon-parametrictestofKruskal–WallisfollowedbyDunn’sposttestonlydetectedsignificantdifferencesbetweenliverandspleenandbetweenliverandstomachs(p < 0.05).

Discussion

TheexponentialcharacterofPCRamplificationmaycompromisequantitativeassaysbecauseitmultipliesvariations.ThecompetitivePCRstrategyusedinthepresentstudywasaimedtoovercomesomeofthelimitationsoftheconventionalRT-PCR.Co-amplificationmethodsquantifythetargetDNArelativetoasecondcontrolsequenceinthesamePCRtube.Themainadvantagesofthistechniquearethattheresultsarenotaffectedbytube-to-tubevariationsinamplificationefficiencyanditisnotnecessarytorestrictPCRtotheexponentialphase.ReliablequantificationisstillpossibleifthePCRextendsintothelinearphase,oreveninthesaturationphase,provideditisascertainedthattheamplificationefficiencyisthesameforbothtemplatesthroughoutthePCR,includingthefinalcycles(12,16).Quantitativeco-amplificationrestsontheassumptionthattheproductratiooftargetandcompetitorsequencesreliablyreflectstheratiooftheirinitialcopynumbers.Therefore,itisrequisitethatefficiencyisidenticalforbothsequences.Ifthetargetsequence(T)andthecompetitorsequence(C)wouldamplifywiththesmallestdifferenceinthisefficiencies,itcanleadtoverydifferentquantitiesoftheendproducts.Thiscanresultinanerroneousestimationoftheamountofinitialmaterial[12,16].Figure3showsthattheefficienciesinoursystemfortargetandcompetitorareequalineachcycle,eveniftheefficienciesdecreaseinthelatercycles.ThevalidityofthecompetitionreactionstoeachquantityoftargetwasestablishedbythegeneratedregressionequationswiththeircorrespondingsignificantR²values(Figure4).Theslopeoftheregressioncurvesobtainedwascloseto1inallfivestandardcurves,indicatingthatnodifferentialamplificationratesexistbetweenTandCintherangeofassayedT.Variabilityinthe10,000-to1,000,000-moleculerangesoftargetsequencewasdetermined.Coefficientsofvariationaccordingtononlog-transformedabsolutevalueswere8%inter-assayandrangingbetween3.14%and8.4%intra-assayfor100,000moleculesofT.CVsof21.89%,36%,12.07%,and13.78%intra-assayfor10,000,150,000,200,000,and1,000,000moleculesofT,respectivelywereobtained(Table3).Ingeneral,lowertargetquantitiesgivehigherCVs.OurvariabilityisintherangeofthevariabilityofthePCRassay[17,18].Ingeneral,CVscorrespondingtoreactionconditions,inwhichthequantityoftargetandcompetitorwasequivalentornearby,wereunder10%,andCVscorrespondingtoreactionconditions,inwhichtheratioC/Twas10or1/10,werebetween10%and35%.Otherauthorsaffirmthatmoretime-consumingmethodsofRNAseprotectionassayandNorthernblotsaremoreaccurateandprecisethanRT-PCR.However,ithasbeenreportedthatRNAseprotectionassaycoulddetectapproximately1×106targettranscriptsandithasbeenestimatedthataNorthernblotisabouttenfoldlesssensitivethanRNAseprotectionassay(19),thentheyarelimitedtostudythosegenesthatarerelativelyhighlyexpressed.OurcompetitivePCRwasabletodetectatleast750moleculesofDNAtargetsequencein50µlofPCR.Ifweassume10%ofefficiencyoftheRTreaction,theLLQofoursystemwouldbe7.5 × 10³targettranscripts.Therefore,thisassayismoresensitivethanRNaseprotectionandNorthernblotassays.OurresultsshowthatourdesignofcompetitivePCRtogetherwithRTreactionisusefultostudytheexpressionleveloftiGHRIindifferenttissuesoftilapia(O.niloticus).ByusinginvitrotranscribedcompetitorRNA,wehavebeenabletoreducesourcesofvariationsuchasthevariableefficiencyofthereversetranscriptionreactionbecausethequantityofcompetitorRNAmoleculesthatweputtogetherwithspecifictissuetotalRNAsamplesisreversetranscribedwiththesameefficiencythatthemoleculesofRNAtargetineachsample.

Wehavealsobeenabletodetectexpressionofthisreceptorinallstudiedtissues,whichisconsistentwiththepleiotropicnatureofgrowthhormoneinfish(20–23).TheexpressionleveloftiGHRthatweobtainedforeachstudiedtissuecanbeorganizedindecreasedorderofexpressionlevelsas:liver > muscle > brain > heart > gonads > intestine > stomach > spleen(Fig.6).ThehighestexpressionleveloftiGHRIinliverisconsistentwithpreviousreceptorbindingstudies(20).Besides,itisinagreementwithpreviousconventionalRT-PCRstudies(4,6)andwithreal-timePCRstudies(24).Asitisexpected,thetissuedistributionobtainedforusismoresimilartothereal-timeRT-PCRresultsthantotheresultsoftheotherstudiesusingconventionalRT-PCR.Thefactthatweobservedstatisticallysignificantdifferencesonlybetweenliver–spleenandbetweenliver–stomachsisduetoanon-parametrictestthatweused.ThesetestsarelesspowerfulthantheparametricteststhatassumedataGaussiandistributions.Withsmallsamples(n = 3),non-parametrictestshavelittlepowertodetectdifferencesespeciallywhenweworkwithBIOLOGicalsamplesthatareintrinsicallyvariable.Infutureexperiments,wewillworkwithahighernumberofanimals.Torefineourinitiallyfoundresults,wewouldretestthemwithahigherresolutionofcompetitormoleculesbecausewedemonstrateinthevalidationexperimentsthatthecoefficientsofvariabilityarelowerwhenthemoleculenumbersoftargetandcompetitorsequencesinthesamplesaresimilar.

DespitethefactthatthedescribedcompetitiveRT-PCRassayislaborintensive,lesssensitive,andhasalowerdynamicrangethanthereal-timeassays,itislessexpensivethanthereal-timeRT-PCRstudies.

Insummary,throughthiswork,wehavedevelopedaquantitativeRT-PCRassaybycompetitionthatwassensitiveenoughtodifferentiateamongmRNAabundancelevelsoftiGHRI.ThenatureofcompetitionreactionsobservedwassupportivetoprovetheauthenticityofquantificationoftiGHRI.

AcknowledgmentsThisresearchwasfundedbytheCenterforGeneticEngineeringandBiotechnology.TheauthorswouldliketothankDr.RicardoL.LleonartforhisvaluableassistanceandreviewofthismanuscriptandMSc.YoelysCruzforheradviceinstatisticalprocessingofdata.

Appendix

PROTOCOLS

Expectedmoleculestarget	1,000,000	200,000	150,000	100,000^a			10,000
Expectedmoleculestarget	1,000,000	200,000	150,000	A	B	C	10,000
Observedmoleculestarget^b	1,173,333	290,000	255,000	115,000	110,667	97,333	11,200
SD	161,658	35,000	91,788	5,000	9,292	3,055	2,081
CV	13.78%	12.07%	36%	4.35%	8.40%	3.14%	21.89%

I-Cloningofcompetitor

Materials

•	RNAgents:TotalRNAIsolationSystem,PromegaZ5110
•	PolyATract:mRNAIsolationSystemII,PromegaZ5200
•	ReverseTranscriptionSystem,PromegaA3500
•	PCRMasterMix,PromegaM7505
•	QIAquickGelExtractionKit,QIAGEN28704
•	pGEM®-TEasyVectorSystemI,PromegaA1360andpBluescript®IIPhagemidVectors,Stratagene212207tocloningDNAsequences
•	Reagentsforcloning
•	Ampicillinandstreptomycinforselectionpurposes
•	DegeneratedprimerstoamplifytiGHRIprobesequence
•	Top10(F⁻mcrAΔ[mrr-hsdRMS-mcrBC]ø80lacZΔM15ΔlacX74deoRrecA1araD139Δ[ara-leu]7697galVgalKrpsL[StrR]endA1nupG)orequivalentelectrocompetentE.colicells
•	T7RiboMAXTMExpressRNAiSystem,PromegaP1700
•	MEGAscript®RNAiKit,Ambion1626

Methods

1.	ToobtaintotalRNAoftilapia(O.niloticus)liver,wefollowedtheproceduredescribedinthesectionIVoftheTechnicalBulletin087(TB087)ofRNAgents®TotalRNAIsolationSystem(Promega).Westartedwith1goftilapialiver.
2.	Startingwith5mgoftilapialivertotalRNA,weobtainedmRNAfollowingexactlytheprotocoldescribedinthesectionIVintheTechnicalManual021(TM021,Promega)toPolyAtract®SystemsI.
3.	RT-PCRamplifiesthetiGHRIprobe.Thereversetranscriptionreactionwasperformedwith1µgoftilapialivermRNAandOligo(dT)₁₅asprimerfollowingtheprotocoldescribedinthesectionIIIoftheTechnicalBulletin099(TB099)ofReverseTranscriptionSystem(Promega,USA).Weused10µlofthefivetimesdilutedRTreactionin50µlofthePCRfinal,volume.Weusedtoo25µlofPCRMasterMix2×(Promega)and3µMofeachdegeneratedprimer(150pmol/50µlPCR).Weperformed3minto95°CtodenaturalizeallDNAsinthereactionandafterwedidcyclingprogram(1minto94°C,1minto42°C,and1minto72°C)for30cyclesandafinalextension5minto72°C.
4.	ClonethePCRproductnearbyto500bpintothepGEM-TEasy,orequivalent,vectorandtransformintoelectrocompetenttoptenE.colicells,orequivalent,forsequenceverification.
5.	Sub-clonethe3′regionofthetiGHRIprobeintopBSKS + vectorandensembleagainduplicatinganinternalPstI–AccIfragment(Fig.1a).
6.	LinearizetheplasmidthatcontainsthecompetitorsequenceunderthecontrolofT7RNApolymerasepromoterwithappropriatedrestrictionenzyme.
7.	Synthesizesingle-strandedtranscriptofcompetitorRNA.WefollowedtheprotocoldescribedinthesectionIII-CoftheTechnicalBulletin316oftheT7RiboMax™ExpressRNAiSystem(Promega)
8.	RemovetheDNAtemplatebydigestionwithRNase-freeDNaseoftheT7RiboMax™ExpressRNAiSystem.
9.	PurificationofssRNAwascarriedoutfollowingtheprotocoldescribedinthesectionIII-EoftheInstructionManualoftheMEGAscript®RNAiKit(Ambion,USA).
10.	Quantitatetheproductbymeasuringitsabsorbanceat260nmandexaminetheintegrityona1%denaturingagarosegel.
11.	StoreRNAcompetitorprecipitatedinEtOHat−20°C.

II.ValidationofCompetitivePCR

Materials

•	LinearDNAoftargetandcompetitorsequences.
•	Gene-specificprimerstoamplifytargetandcompetitorsequence.
•	PCRMas,terMix,PromegaM7505
•	ReagentsandequipmenttodoDNAelectrophoresis.
•	DigitalcameraOlympusC7070WideZoom
•	ImageJprogram(Version1.33).

Methods

DeterminationofequalPCRamplificationefficiencyoftargetandcompetitorsequences.

a.	EnsembletenidenticalPCRtubescontainingequimolecularquantitiesoftargetandcompetitorsequencesin50µlasfinalvolume.Use0.2µMofeachspecificprimerand25µlofPCRMasterMix.Perform3minto95°CtodenatureallDNAandacyclingprogram30sto94°C,30stoannealingtemperatureaccordingtheTmofthespecificoligonucleotides,and1minto72°C.Removeonetubeat17,19,21,24,27,30,33,36,39,and45cycles,respectively,andstoreat−20°C.
b.	Electrophoresisin2%agarosegelinTA1×(0.04MTris–acetate,0.001MEDTA,pH7)oftheamplificationproductsofeachtube.
c.	Takethedigitalimagesofthegel.
d.	DensitometryanalysisofcompetitorandtargetbandsineachlaneusingImageJProgram(Version1.33).Thisprogramisidealtocomparebandsinthesamedigitalimage.Itisbasedonthefactthatopticaldensity(OD)isalogarithmicfunctionofbrightness.AsetofmacrosarebundledtoImageJwhichareusedforgeldensitometryanalysisproducingcurves.Theheightofthecurve,atanygivenpoint,isthemeanoftheODofagivenrowofpixelsinthemarkedlane.Theprogramcancalculateareaofuser-definedselections.TheareameasurementsarerecordedintabularformandaredisplayedinaResultswindowasshowninFig.7.Togetmoreinformation,visithttp://rsb.info.nih.gov/ij/
e.	Plottheareameasurementsversuscyclenumbertotargetandcompetitor,respectively(Fig.3).
f.	CalculateefficiencytoeachcycletotargetandcompetitoraswasdescribedinSection2.
g.	Comparetheefficienciesoftargetandcompetitorusingapairedttest.
h.	Accordingtotheresults,establish30cyclesforallcompetitivePCRs.

Determinationofthesensibilityofthismethod.

EnsemblePCRsinthesameconditionsestablishedbeforeusingdecreasedquantitiesoftargetwithdifferentdilutionsofcompetitorwithreplicasforeachpoint.Inourexperiment,weensemble:

•	2,000moleculesoftargetwith750,1,000,and5,000moleculesofcompetitor,respectively.
•	1,000moleculesoftargetwith500,1,000,and5,000moleculesofcompetitor,respectively.
•	750moleculesoftargetwith,250,750,and1,000moleculesofcompetitor,respectively.
•	100moleculesoftargetwith50,100,and500moleculesofcompetitor,respectively.
•	10moleculesoftargetwith5,10,and50moleculesofcompetitor,respectively.

Electrophoresisin2%agarosegelinTA1×(0.04MTris–acetate,0.001MEDTA,pH7)oftheamplificationproductsofeachtube.

Determinationofminimumquantityoftargetthatthemethodisabletodetect.

Characterizationofthemethodprecisionandrepeatability.

EnsemblePCRsinthesameconditionsestablishedbeforeusingquantitiesoftargetintherangeofyourexpectedmeasurementswithdifferentdilutionsofcompetitorwithreplicasforeachpoint.Inourexperiment,weensemble:

•	10⁴moleculesofTwith5×10³,10⁴,5×10⁴,7.5×10⁴,and10⁵moleculesofC,respectively,withthreereplicasforeachpoint.
•	10⁵moleculesofTwith10⁴,5×10⁴,7.5×10⁴,10⁵,5×10⁵,and7.5×10⁵moleculesofC,respectively,withthreereplicasforeachpoint.Thisexperimentwasrepeatedthreetimesindifferentdaystoassaytheinter-experimentvariability.
•	1.5×10⁵moleculesofTwiththesamemoleculequantitiesofCasthepreviousexperimentwiththreereplicasforeachpoint.
•	2×10⁵moleculesofTwith10³,10⁴,5×10⁴,10⁵,5×10⁵,and10⁶moleculesofC,respectively,withthreereplicasforeachpoint.
•	10⁶moleculesofTwith5×10⁵,10⁶,5×10⁶,and10⁷ofC,respectively,withthreereplicasforeachpoint.

Electrophoresisin2%agarosegelinTA1×(0.04MTris–acetate,0.001MEDTA,pH7)oftheamplificationproductsofeachtube.

Takedigitalimagesofthegel.

DensitometryanalysisofcompetitorandtargetbandsineachlaneusingImageJProgram(Version1.33)aswasdescribedinpoint1dofthissection.

MakelinealregressionsforeachexperimentplottinglogoftheratiobetweentheareameasurementsofCandTversuslogoftheCmoleculenumbers.DeterminetheequationofeachregressionwithitsrespectiveR2.

Determinethexvalue(Cmoleculenumber)wheny=0.ThisconditionoccurswhentheareameasurementsofCandTareequalbecauseC/T=1andthelog1=0.Then,xvalueisequaltothecalculatedTmolecularnumberineachexperiment.

PlotTmoleculenumbersexpectedversusobservedanddeterminethecorrelationcoefficient(r).WeusedthetestofPearsoncorrelation.ThisdeterminationrepresentsameasureoftheprecisionofourmethodintherangeoftheTquantitiesassayed.

Calculatethevariationcoefficients(CVs)intra-experimentusingthereplicasinsideexperimentsandinter-experiments.TheseCVvaluescharacterizetherepeatabilityofourmethodintherangeoftheTquantitiesassayed.

III.CompetitiveRT-PCR

Materials

•	RNAgents:TotalRNAIsolationSystem,PromegaZ5110
•	CompetitorRNAgeneratedintheProtocolI
•	Gene-specificprimerstoamplifytargetandcompetitorsequence.
•	Ready-to-GoRT-PCRBeads,AmershamBiosciences,27-9259-01
•	ReagentsandequipmenttodoDNAelectrophoresis.
•	DigitalcameraOlympusC7070WideZoom
•	ImageJprogram(Version1.33).

Methods

1.	IsolatetotalRNAofeightdifferenttissues(brain,muscle,heart,liver,gonads,stomach,spleen,andintestine)fromthreetilapias(O.niloticus).TotalRNAmini-preparationsstartingwith50mgofeachtissuewereperformedusingRNAgents.TotalRNAIsolationSystem,Promega.(seetable1oftheTB087).
2.	EnsemblereactionsofRT-PCRusingReady-to-GoRT-PCRBeads,AmershamBiosciences.Weused2µlofeachpreparationoftotaltissue-specificRNAwithatleastthreeknowndifferentquantitiesofcompetitorRNAandrandomprimerstotheRTreactionsand0.2µMofeachspecificprimertothePCR.
3.	Electrophoresisin2%agarosegelinTA1×(0.04MTris–acetate,0.001MEDTA,pH7)oftheamplificationproductsofeachtube.
4.	Takedigitalimagesofthegel.
5.	DensitometryanalysisofcompetitorandtargetbandsineachlaneusingImageJProgram(Version1.33)aswasdescribedinpoint1dofthissection.
6.	DolinealregressionsforeachexperimentplottinglogoftheratiobetweentheareameasurementsofCandTversuslogoftheCmoleculenumbers.Determinetheequationofeachregression.
7.	Determinethexvalue(Cmoleculenumber)whenyisequalto0.Inthiscondition,xvalueisequaltoTmoleculenumberineachRNAsample.
8.	NormalizethisTmoleculenumberineachRNAsamplewiththeinputoftotalRNAintheRTreaction.ThetotalRNAinputwasdeterminedbydensitometryanalysisof28SsubunitofribosomalRNAineachsampleusingImageJprogram.Wedidanelectrophoresison1.2%agarosedenaturalizinggelof2µlofeachtotalRNApreparation.Thedensitometrydataofthebandscorrespondingtothe28SsubunitsmeasuredwithImageJprogramwereconvertedtomicrogramsoftotalRNAusingareferenceRNAwithknownconcentration.Then,theresultswereexpressedastiGHRImoleculenumbers/µgRNAt.
9.	AveragethetiGHRImoleculenumbers/µgRNAtamongthethreetilapiastoeachtissue.Theseaverageswerecomparedbynon-parametricKruskal–WallistestfollowedbyDunnmultiplecomparisontest.

MediaObjects/12575_2009_9002_Fig7_HTML.gif

Fig.7DensitometryanalysisofcompetitorandtargetbandsusingImageJProgram.aUser-definedselectionfrom2%agarosegeldigitalimage.bImageJplotsofselectedimage.cTheareameasurementsdisplayedinaResultswindowbytheimageJProgram.