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Methods to Remove DNA Contamination from RNA Samples
蚂蚁淘
/发布时间:1545759430 /浏览量:141
AfrequentcauseofconcernamonginvestigatorsperformingquantitativeRT-PCRisfalsepositivescausedbygenomicDNAcontaminationofRNApreparations.BecausePCRissuchasensitivetechnique,asinglecopyofagenecan,theoretically,bedetected.HerewetestvariousmethodsforremovingDNAcontamination.GenomicDNAfalsepositivesignalsareeasilyidentifiedbyperforminga"no-RT"controlduringRT-PCR.WecanthereforeassesstheeffectivenessofDNAremovalmethodsbyagarosegelanalysisofthe-RTreactions. ThreeMethodsofRemovingContaminationfromRNA ThedatapresentedhereresultsfromtestingseveralcommonmethodsofDNAremovalfromRNAsamples.EachDNAremovaltechniquewasanalyzedforeffectivenessbyPCRamplificationoftheRNAwithandwithoutpriorreversetranscription.Themethodsinclude: DNasedigestionDNaseisanendonucleasethatcleavesDNAbybreakingphosphodiesterbonds.ItmustbeinactivatedorremovedfromthereactionpriortoPCR,otherwise,itmaydigestnewlyamplifiedDNA.Forthisstudy,wetested2concentrationsofDNaseI(10and50µlDNaseI/mlsample)andfourDNaseIinactivation/removalmethods: Acidphenol:chloroformextractionAcidphenol:chloroform(5:1phenol:CHCl3;pH4.7)extractionpartitionsDNAintotheorganicphase.TheRNAremainsintheaqueousphaseandcanbesubsequentlyrecoveredbyprecipitation. Lithiumchloride(LiCl)precipitationLiClprecipitationisaselectiveprecipitantofRNA.ItinefficientlyprecipitatesDNAwhichisdiscardedinthesupernatant. AssessingDNAContaminationofRNASamples ToconfirmthepresenceofcontaminatingDNA,twoRNAsampleswereassessedona1%denaturingagarosegelbyethidiumbromidestaining(Figure1a).OneRNAsampleshowedvisIBLeDNAcontamination(sampleB),whiletheotherdidnot(sampleA).ThesamesampleswerethensubjectedtoRT-PCR,alongwitha"no-RT"control,forthepresenceofribosomalproteinS15message.RegardlessofwhethercontaminatingDNAwasvisiblebygelassessment,bothRNAsamplesshowedamplifiableDNAintheno-RTcontrol(Figure1b).OnceDNAcontaminationwasverified,bothRNAsamplesweresubjectedtothethreeDNAremovalmethodsdescribedabove. Figure1b.1µgofeachRNAfromFigure1awasusedinareversetranscriptionreactionperformedwiththeRETROscriptÌKit.1µlofeachRTreaction(RT-PCR)and0.5µgofeachRNA("no-RT"control)weresubsequentlyusedinastandardPCRreactionfortheamplificationoftheribosomalproteinS15message.One-tenthofeachPCRreactionwasassessedona1%agarosegelandstainedwithEtBr. Results DNasedigestionBothRNAsamplesA&Bweretreatedwith10and50UDNaseI(Ambion,Inc.)permlRNAsampleat37°Cfor30minutes.ThedigestedsamplesweresplitintofourtubesandeachwastreatedwithadifferentDNaseinactivationorremovalmethod.ThedatarevealthatDNasetreatmentfollowedbyanyoftheinactivationmethodswassufficienttoremovecontaminatingDNAastestedbytheno-RTcontrol(Figure2a)anddidnotinhibitsubsequentRT-PCR(Figure2b). Acidphenol:chloroformextractionAliquotsofeachRNAsamplewereextractedwithanequalvolumeofacidphenol:chloroformfollowedbyprecipitationwith0.5MNH4OAcandEtOH.TheRNApelletswereresUSPendedinnuclease-freewaterandthentestedinRT-PCRreactions.AswithDNasedigestion,acidphenol:chloroformextractionremovedcontaminatingDNAwithouteffectingRT-PCR. LiClprecipitationRNAsamplesAandBwereprecipitatedwithequalvolumesof7.5MLiCl,resuspendedinnuclease-freewaterandusedinRT-PCRreactions.LiClprecipitationdidremoveDNAfromsampleAbutwasinsufficienttoremovethegreaterDNAcontaminationinRNAsampleB(Figures2aand2b). Figure2b.Approximately1µgofeachtreatedRNAwasusedinaRT-PCRreactionfortheamplificationofribosomalproteinS15messageusingAmbion"sRETROscriptÌKit.One-tenthofeachRTPCRreactionwasassessedona1%agarosegelandstainedwithEtBr. Conclusions ThreecommonmethodsusedtoremovecontaminatingDNAfromRNApreparationsweretested.ThemethodsincludedDNaseIdigestion,acidphenol:chloroformextractionandLiClprecipitation.BothDNaseIdigestion(withsubsequentDNaseIinactivationorremoval)andacidphenol:chloroformextractionweresufficienttoremovecontaminatingDNAastestedbyno-RTcontrolPCRreactions.ThesemethodsworkedwellevenwhentheamountofDNAcontaminationwasvisiblebyEtBrstainingoftheRNAsample(sampleB).NeithermethodinhibitedsubsequentRT-PCRreactions.LiClprecipitationasamethodtoremovecontaminationDNAfromRNAsampleswasnotasefficient.LiClprecipitationwasadequatetoremovemoderatebutnotgrossDNAcontaminationfromRNAsamples.LiClprecipitationalsodidnotinhibitsubsequentRT-PCRreactions.
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Figure1.AssessmentofDNAContaminationinRNAPreparations.Figure1a.1µgofeachoftwoRNAsamples(labeledAandB)alongwith3µgofAmbion"sMillenniumÌMarkerswasassessedona1%denaturingagarosegelandstainedwithEtBr.NoticeinSampleB,thehighmolecularweightDNAcontamination. 
Figure2.AssessmentofMethodstoremoveDNAContaminationofRNA.ThetwoRNAsamplesshowninFigure1aweretreatedwithDNase,extractedwithacidphenolorprecipitatedwithLiClasamethodofDNAremoval.TheRNaseweretreatedwitheither10Uor50UDNaseI/mlRNAsample,splitintofouraliquotsandtheDNaseIwasinactivatedbyoneoffourmethods: Samples:
Figure2a.Approximately0.5µgofeachtreatedRNAwasusedinastandardPCRreactionfortheamplificationofribosomalproteinS15message.One-tenthofeachPCRreactionwasassessedona1%agarosegelandstainedwithEtBr.
