Figure1.DNAContaminationinRNAIsolatedbyFiveDifferentMethods.MouselivertotalRNAwasisolatedaccordingtoprotocolbyfivedifferentmethods.0.5µgRNAwasusedinRT-PCRreactionswithAmbion"sRETROscript?Kit.PCRreactionswereperformedwith5µgRNA.10µlofeachreactionwaselectrophoresedona2%agarosegelandstainedwithEtBr.

DifferentialEnrichmentbyOligod(T)Selection

Althoughtworoundsofoligod(T)selectionaresufficienttoremovegenomicDNAcontamination,therearetwodrawbackstousingthistechniquetocontrolforDNAcontamination.First,oligod(T)chromatographyisexpensiveandlaborintensiveforroutineanalysis.Secondly,apotentiallyseriousproblemnotusuallyaddressedisthatrelativeamountsofindividualtranscriptscanchangewitholigod(T)chromatography,probablyasaresultofdifferentialpolyadenylationbetweentissuesorinresponsetostimuli.AtAmbion,wehavefoundthatoligod(T)selectioncanevenchangetheapparentabundanceoftranscriptsfromgenesthatarethoughttohaveinvariantexpression.Forexample,whenwecomparetherelativeenrichmentofcyclophilinandGAPDHtranscriptsbyNorthernblotanalysisoftotalversusoligod(T)selectedmouseRNA,weseeanobviouschangeintheapparentabundanceofthesetwotranscripts.AsshowninFigure2,oligod(T)selectionenrichesGAPDHandcyclophilin17Xand22X,respectively,fromkidneyRNA,but21Xand28XfromthymusRNA.Thesourceofthisvariationisunclear,buttheimplicationsforquantitationfromoligod(T)selectedRNAareimpossIBLetoignore.

Figure2.DifferentialEnrichmentofSpecificmRNAsbyOligodTChromatography.ANorthernblotcontainingtotalRNA(1µg)andtwiceoligod(T)selectedRNA(50ng)frommousethymusandkidneywashybridizedsimultaneouslywithGAPDHandcyclophilinRNAprobes.HybridizationsignalswerequantitatedwithaBio-RadMolecularImager.

PrimerDesign

Primersforquantitativeexperimentsaretypicallydesignedtoamplifyatargetbetween150and600basepairs.Targetssmallerthan200bparedifficulttoresolveonagarosegels,andlargertargetsplaceagreaterburdenontheinvestigatortooptimizePCRconditions.ThecriticalaspectforRT-PCRprimerchoicewithrespecttominimizingtheproblemsassociatedwithDNAcontaminationistodesignprimersthatspanatleastoneintronofthegenomicsequence.ThiswillresultinaPCRproductfromgenomiccontaminationthatwillbelargerinsizethantheproductgeneratedfromthecDNA.Infact,primerscanbedesignedtospanasufficientlylargegenomicfragmentsuchthatamplificationfromcontaminatingDNAmaybenotbepossible.Forgenesinwhichthegenomicsequenceispublished,thepositionsofthesplicejunctionscanbefoundbyretrievingthesequencefromtheGenbankdatabaseathttp://www.ncbi.nlm.nih.gov/Genbank/index.html.Iftheintron-exonstructureisunknown,primerscanbesynthesizedindifferentregionsofthecDNAsequenceandtriedincombinationsonbothcDNAandgenomicDNA.Itshouldbepossibletochooseaprimercombinationthatyieldseithernoproduct(additionalintronsequenceproducestoolongatargetforefficientPCR)oraneasilydistinguishableproductwhenamplifyingfromgenomicDNA.Anadditionalwrinkleisthatpseudogenesexistinthemammaliangenomeformanygenes,includingthemostcommonlyusedinternalcontrols(ß-actin,GAPDH,cyclophilin).Thesesequences,arisingfromintegrationofareversetranscriptionproductintothegenome,donothaveintrons.Thus,thesizeofaPCRproductamplifiedfromapseudogenemaybeidenticaltothatproducedfromacDNAcopy.Theonlywaytoidentifytheseproductsistoperforma"no-RT"controlasshowninFigure3.ThetrueproductfromRNAis361basepairs.The"no-RT"controlyieldsbothafragmentidenticalinsizetotheexpectedRT-PCRproductfromtheRNAtranscript(fromapseudogene),anda1.2kbfragmentfromthelegitimategenomiclocus.Ifitisabsolutelyessentialtoavoidamplificationfromthesesequences,anamplifiedfragmentfromapseudogenemaybesequenced,andprimersdesignedtoregionswherethesequencevariesfromthelegitimatecopyofthegene.Aslittleasaoneortwonucleotidedifferenceatthe3"endofaprimerbindingsitecancompletelyinhibitamplificationfromthepseudogene.

Figure3.DNAContaminationinRNA.MouselivertotalRNAwasisolatedaccordingtoprotocol.RT-PCRreactionswereperformedusingAmbion"sRETROscript?Kitand0.5µgRNA.PCRreactionswereperformedwith5µgRNA.10µlofeachreactionwaselectrophoresedona2%agarosegelandstainedwithEtBr.

DNaseITreatment

InarecentinformalsurveyofRT-PCRusers,wefoundthatthefieldisevenlydividedbythoseuserswhobelievethatDNaseItreatmentsolvestheproblemofgenomicDNAcontaminationandthosewhofeelthatDNaseItreatmentisanunacceptablesolution.DetractorsclaimthatDNaseItreatmentandthesubsequentinactivationstepscompromisetheperformanceoftheirRT-PCRreactionstoanunacceptabledegree.MuchoftheproblemtheseusersexperiencemaybetracedtotheextremetemperaturesusedtoinactivatetheDNaseIpriortoreversetranscription.Huang,etal.(Biotechniques,(1996)20:(6)1012-1020)reportcompleteinactivationofDNaseIbyheatdenaturationat75°Cfor5minutes.LowerinactivationtemperaturesdonotcompletelyinactivateDNaseI,whilehighertemperaturesappeartodamagetheRNAtemplate.DNaseItreatmentfollowedbyheatinactivationisasimpleenoughtechniqueforroutineuseinsystemsinwhichgenomicDNAcontaminationisaproblem.Theuseofhighquality,RNase-freeDNaseiscrucial.TwoadditionalconventionalmethodsofreducingcontaminatinggenomicDNAfromtotalRNApreparationsareacidphenolextraction,whichpartitionsDNAintotheorganicphase,andLiClprecipitation,whichselectivelyprecipitatesRNAfromsolution(proteinandDNAremaininthesupernatant).AdescriptionofthesetechniquescanbefoundinAmbion"sTechnicalBulletins#158and#160.ThesetechniquescanbeusedafterDNaseItreatmenttoinactivatetheenzymeandprecipitatetheRNApriortoreversetranscription.Finally,itshouldbenotedthatDNaseItreatmentneitherrelievestheinvestigatoroftheburdenofsensibleprimerdesign,norofthenecessitytoperformtheappropriate"no-RT"controls.

Inadditiontotheabovetechniques,researchersnowhaveanewandconvenientoptionforremovalofDNAandDNaseIfromRNAsamples.DNA-free™DNaseTreatmentandRemovalReagentsaredesignedfortheremovalofcontaminatingDNAfromRNAsamplesandfortheremovalofDNaseaftertreatment.Asdescribedabove,DNaseistypicallyinactivatedbyheattreatment,andcanalsoberemovedfromtreatedprepsbyphenolextraction.Heatinactivationcanpresentproblems,however,asthetemperatureatwhichDNaseisinactivatedalsocatalyzesRNase-independentRNAstrandscissioninthepresenceofdivalentcations.Phenolextractionisalsoavoidedbyresearcherswhodonotwanttoworkwithphenol,orwhoworryaboutsampleloss.

DNA-freeavoidsbothmethodsofDNaseIinactivationbysupplyinganovelDNaseRemovalreagentthateffectivelyremovesDNaseanddivalentcationsfromthereactionmixture.TheDNase/cationremovalsteptakesamerethree-minuteincubation.Noorganicextraction,EDTAadditionorheatinactivationisrequired.

TheDNA-freeDNaseTreatmentandRemovalReagentsareprovidedwithRNase-freeDNaseI,anoptimized10XReactionBuffer,andtheDNaseRemovalReagent.TheDNA-freeReagentsarenowalsopartoftheRNAqueous™-4PCRKit,combiningthefeaturesandbenefitsofRNAqueous™withthoseofDNA-free.

Figure4.RT-PCRExperimentsUsingTotalRNADNase-TreatedUsingDNA-freeReagents.FiveµlofRNAsamplesisolatedusingAmbion"sRNAqueous™Kitwereusedastemplatesforreversetranscriptionreactions;10%oftheresultingcDNAwasamplifiedbyPCRusingS15primers.ThelanestotheleftoftheMarkersarePCRreactionsdonewithoutreversetranscription,demostratingthelackofgenomicDNAcontaminationintheseRNAsamples.ThelanestotherightofthemarkersshowtheS15RT-PCRproductfromtheindicatedsamples.

InadditiontoDNA-free,AmbionoffersmanyqualityproductstofacilitatesuccessfulRT-PCRexperiments.TheseincludeRNase-freePipettetipsandPCRtubes,RNasefreeDNaseI,ToTALLYRNA,RNAqueous,andPoly(A)PureRNAIsolationKits,RETROscriptFirstStrandcDNASynthesisKit,andSuperTaq?ThermostableDNApolymerase.AllofAmbion"sproductsdesignedforusewithRNAundergorigorousqualitytestingandarecertifiedRNase-free.

ThePolymeraseChainReaction(PCR)iscoveredbypatentsownedbyHoffman-LaRoche.UseofthePCRprocessrequiresalicense.AlicenseforresearchmaybeobtainedbypurchaseanduseofauthorizedreagentsandDNAthermalcyclers.

SuperTaq™ismadebyEnzymeTechnologiesLimitedandsoldunderlicensingarrangementswithF.Hoffmann-LaRocheLtd.,RocheMolecularSystems,Inc.andthePerkin-ElmerCorporation.AmbionisadistributorofEnzymeTechnologiesLimited.

PurchaseofSuperTaqisaccompaniedbyalimitedlicenseforitsuseinthePolymeraseChainReaction(PCR)andRT-PCRprocessforresearchinconjunctionwithathermalcycler,theuseofwhichintheautomatedperformanceofthePCRandRT-PCRprocessiscoveredbytheup-frontlicensefee,eitherbypaymenttoPerkin-Elmer,oraspurchased,i.e.,anauthorizedthermalcyler.

SuperTaqisnotavailableforsaledirectlyfromAmbionintheUnitedKingdom,France,BeNeLux,Denmark,Sweden,Italy,Austria,Switzerland,Singapore,andTaiwan.ContactEnzymeTechnologiesLTD,Unit4,61DittonWalk,CambridgeCB58QD,U.K.(phone44-1223-412-583)formoreinformation.