The Basics: In Vitro Transcription | Thermo Fisher Scientific - TW

Such probes are much more sensitive than random-primed DNA probes. Small scale reactions may also be used to synthesize RNA transcripts containing modified ... PopularApplications&TechniquesShopAllProductsServicesSupport Signin QuickOrder     Search ThermoFisherScientific SearchAll Search Home›TechnicalReferenceLibrary›RNATechnicalResourcesfromAmbion›ProbeLabelingSystems›GeneralArticles›TheBasics:InVitroTranscriptionTheBasics:InVitroTranscriptionSeeNavigation‹GeneralArticlesAmbion'sBrightStarNonisotopicDetectionSystem ›AminoAllylLabelingforArrayAnalysis ›BrightStarPsoralen-BiotinLabeledProbes ›MethodsforEnzymaticNonisotopicLabelingofRNAbyinVitroTranscription ›NonisotopicDetection ›ProbeLabelingChart ›ProbeSpecificActivity ›TheBasics:InVitroTranscription ›WhenLabeledNucleotidesGoBad ›TheabilitytosynthesizeRNAinthelaboratoryiscriticaltomanytechniques.RadiolabeledandnonisotopicallylabeledRNAprobes,generatedinsmallscaletranscriptionreactions,canbeusedinblothybridizationsandnucleaseprotectionassays.Suchprobesaremuchmoresensitivethanrandom-primedDNAprobes.SmallscalereactionsmayalsobeusedtosynthesizeRNAtranscriptscontainingmodifiednucleotidesforvariousbiochemicalandmolecularbiologystudies.Largescaletranscriptionreactions,generatingupto200µgofRNAperreactioncanbeusedforaRNAamplification,expressionstudies(microinjection,infectionwithviraltranscripts, invitro translation),structuralanalysis(protein-RNAbinding),andmechanisticstudies(ribozymeanalyses).Inthisarticle,wepresentanoverviewoftranscription,includingrequirementsof invitro transcriptionreactionsandacomparisonofconventionalvs.largescaleRNAsynthesis. Quicklinks RequirementsRNAphagepolymerasesTemplateoptionsSenseandantisenseConventionalorlarge-scale?Products RequirementsfortranscriptionInvitrotranscriptionrequiresapurifiedlinearDNAtemplatecontainingapromoter,ribonucleotidetriphosphates,abuffersystemthatincludesDTTandmagnesiumions,andanappropriatephageRNApolymerase.TheexactconditionsusedinthetranscriptionreactiondependontheamountofRNAneededforaspecificapplication..Top TranslationsystemsRabbitreticulocytelysateRabbitreticulocytelysateisahighlyefficientinvitroeukaryoticproteinsynthesissystemusedfortranslationofexogenousRNAs(eithernaturalorgeneratedinvitro).Invivo,reticulocytesarehighlyspecializedcellsprimarilyresponsibleforthesynthesisofhemoglobin,whichrepresentsmorethan90%oftheproteinmadeinthereticulocyte.Theseimmatureredcellshavealreadylosttheirnuclei,butcontainadequatemRNA,aswellascompletetranslationmachinery,forextensiveglobinsynthesis.TheendogenousglobinmRNAcanbeeliminatedbyincubationwithCa2+-dependentmicrococcalnuclease,whichislaterinactivatedbychelationoftheCa2+byEGTA.WeoffersanInvitrogen™ nuclease-treatedreticulocytelysate.ThistypeoflysateisthemostwidelyusedRNA-dependentcell-freesystembecauseofitslowbackgroundanditsefficientutilizationofexogenousRNAsevenatlowconcentrations(Figure1).Exogenousproteinsaresynthesizedatarateclosetothatobservedinintactreticulocytecells.Figure1.Standardinvitrotranslationprocedureusingrabbitreticulocytelysateorwheatgermextract.UntreatedreticulocytelysatetranslatesendogenousglobinmRNA,exogenousRNAs,orboth.Thistypeoflysateistypicallyusedforstudyingthetranslationmachinery,e.g.,studyingtheeffectsofinhibitorsonglobintranslation.Boththeuntreatedandtreatedrabbitreticulocytelysateshavelownucleaseactivityandarecapableofsynthesizingalargeamountoffull-lengthproduct.BothlysatesareappropriateforthesynthesisoflargerproteinsfromeithercappedoruncappedRNAs(eukaryoticorviral).TopTemplateoptions:plasmids,PCRproducts,oligonuclotidesandcDNA TheDNAtemplatemustcontainadouble-strandedpromoterregionwherethephagepolymerasebindsandinitiatesRNAsynthesis.Transcriptiontemplatesincludeplasmidconstructsengineeredbycloning,cDNAtemplatesgeneratedbyfirst-andsecond-strandsynthesisfromanRNAprecursor(e.g.,aRNAamplification),andlineartemplatesgeneratedbyPCRorbyannealingchemicallysynthesizedoligonucleotides.PlasmidsManycommonplasmidcloningvectorsincludephagepolymerasepromoters.Theyoftencontaintwodistinctpromoters,oneoneachsideofthemultiplecloningsite,allowingtranscriptionofeitherstrandofaninsertedsequence.SuchdualopposablepromotervectorsincludeInvitrogen'spDP,Promega'spGEM,Stratagene'spBluescriptandInvitrogen'spCRIIvectors.Invitrogen™pTRIPLEscript™familyofvectorscontainallthreephagepolymerasepromotersintandem(onthesamesideofthemultiplecloningsite),allowinganyofthethreepolymerases,SP6,T7orT3tobeused.Plasmidvectorsusedastranscriptiontemplatesshouldbelinearizedbyrestrictionenzymedigestion.BecausetranscriptionproceedstotheendoftheDNAtemplate,linearizationensuresthatRNAtranscriptsofadefinedlengthandsequencearegenerated.Therestrictionsiteneednotbeunique,andprovidingthepromoterremainsadjacenttothetranscriptiontemplate,thevectoritselfmaybedigestedmultipletimes.Itisalsounnecessarytopurifythepromoter-insertsequenceawayfromotherfragmentspriortotranscriptionbecauseonlythefragmentcontainingpromotersequencewillserveastemplate.Restrictionenzymedigestionshouldbefollowedbypurificationsincecontaminantsinthedigestionreactionmayinhibittranscription.PCRproductsPCRproductscanalsofunctionastemplatesfortranscription.ApromotercanbeaddedtothePCRproductbyincludingthepromotersequenceatthe5'endofeithertheforwardorreversePCRprimer.Thesebasesbecomedouble-strandedpromotersequenceduringthePCRreaction.OligonucleotidesTwooligonucleotidescanalsobeusedtocreateshorttranscriptiontemplates.Twocomplementaryoligonucleotidescontainingaphagepromotersequencearesimplyannealedtomakeadouble-strandedDNAtemplate.OnlypartoftheDNAtemplate—the-17to+1basesoftheRNApolymerasepromoter—needstobedouble-stranded.Itmaybemoreeconomical,therefore,tosynthesizeoneshortandonelongoligonucleotide,generatinganasymmetrichybrid(see"MinimalSequenceRequirements").cDNAAmorerecentuseofinvitrotranscriptionisinaRNAamplificationreactions.Forthesereactions,transcriptiontemplatesaregeneratedfromRNAbyusinganInvitrogen™ oligo(dT)-T7promoterprimerduringreversetranscription.ThecDNAisconvertedtoadouble-strandedtranscriptiontemplatebyasecond-strandsynthesisreaction.Top Senseorantisense?Whendesigningatranscriptiontemplate,itmustbedecidedwhethersenseorantisensetranscriptsareneeded.IftheRNAistobeusedasaprobeforhybridizationtomessengerRNA(e.g.,Northernblots,insituhybridizations,andnucleaseprotectionassays),complementaryantisensetranscriptsarerequired.Incontrast,sensestrandtranscriptsareusedwhenperformingexpression,structuralorfunctionalstudiesorwhenconstructingastandardcurveforRNAquantitationusinganartificialsensestrandRNA.The+1GoftheRNApolymerasepromotersequenceintheDNAtemplateisthefirstbaseincorporatedintothetranscriptionproduct.TomakesenseRNA,the5'endofthecodingstrandmustbeadjacenttoorjustdownstreamof,the+1Gofthepromoter.ForantisenseRNAtobetranscribedthe5'endofthenoncodingstrandmustbeadjacenttothe+1G.Iftheinsertisinavector,thevectorshouldbelinearizeddownstreamfromthepromoterandtheinsertedsequencetobetranscribed(see"DoesItMakeAntisense?").Top Conventionalorlarge-scalesynthesis?Invitrotranscriptionreactionscanbedividedintotwotypes:conventionalandlargescale.ConventionalreactionsaretypicallyusedforsynthesizingradiolabeledRNAprobesorforincorporatingmodifiednucleotidesintotranscripts.Large-scalereactions,whichgenerate>100µgRNAperreaction,areusefulforstructuralandexpressionstudies,aswellasforaRNAamplification.Conventionalreactions:synthesisoflabeledRNAprobesormodifiedtranscriptsConventionalreactionconditions,suchasthoseusedintheInvitrogen™ MAXIscript™Kit,userelativelylownucleotideconcentrations(0.5mMeach).Highernucleotideconcentrationsarenotnecessarysince,inthesereactions,thelowconcentrationofradiolabeledormodifiednucleotidepresenteffectivelylimitsthetotalyieldofthereaction.Thetotalconcentrationofthelimitingnucleotide(labeled/modifiedandunlabeled)shouldbeatleast3µMforefficientsynthesisoffulllengthRNAtranscriptsof<400nt(morewillbeneededtosynthesizelongertranscripts).A3µMconcentrationofradiolabeledrNTPcanbeobtainedbyadding5µlofa800Ci/mmol,10mCi/ml(or12.5µM)solutionof[α-32P]NTP.HigherspecificactivitylabeledrNTPsareavailable,butareprovidedatamuchlowerstockmolarconcentration(e.g.the3000Ci/mmol,10mCi/mlhasastockconcentrationofonly3.3µM).WithouttheadditionofunlabeledNTP,itisimpossibletoachievethefinalminimum3µMreactionconcentration.Becauselimitingnucleotideconcentrationcanresultinprematureterminationoftranscription,thereisatrade-offbetweensynthesisofhighspecificactivity(orextensivelymodified)transcriptsandfulllengthtranscripts.Dilutingthelimitingradiolabeledormodifiednucleotidewithunlabelednucleotideproportionallylowersthespecificactivity(orextentofmodification)ofthetranscript,butyieldsmorefulllengthtranscript.Tomakeveryhighspecificactivityorextensivelymodifiedtranscriptsoneshouldlimitoromitanyunlabeledlimitingnucleotidepresent.WhentranscribingRNAfromtemplateslackingCTPandTTPinthe12basesimmediatelydownstreamfromthetranscriptionstartsite,the3µMlimitingnucleotideminimumcanbeovercome(1).Invitrogen'sCUMinusPromoterTechnologyprovidesvectorscontainingCTPandTTP-minusRNApolymerasepromotersaswellasconversionprimersthatcanbeusedtoeliminateCTPandTTPbasesfromRNApolymerasepromotersinexistingvectors.Suchtemplatesproduceahighproportionoffulllengthtranscriptsinreactionscontainingaslittleas0.165µMtotallimitingnucleotide.UsingCUMinustechnology,thehighestspecificactivityradiolabelednucleotidesavailablecannowbemadebyinvitrotranscriptionwithoutadditionofunlabelednucleotide.Asaresult,RNAprobeswith7.5Xhigherspecificactivitycanbetranscribed.Large-scalesynthesis:forstructuralandexpressionstudies,andaRNAamplificationLarge-scaleinvitrotranscriptionreactionscanproduceupto120-180µgRNApermicrogramtemplateina20µlreaction.Novel,patentedtechnologydevelopedbyInvitrogen(i.e.,MEGAscript™technology,seebelow)allowsthephageRNApolymerasestoremainactiveathighnucleotideconcentrationsthatwouldordinarilyinhibittheenzyme.Yieldsfromtheselarge-scalereactionsaretypically10to50timeshigherthanthosepossiblewithconventionaltranscriptionreactions(withoutanylimitingnucleotide).Reactionconditions(e.g.,thetypeofnucleotidesalt,typeandconcentrationofsaltinthetranscriptionbuffer,enzymeconcentrationandpH)arealloptimizednotonlyforeachpolymerasebutfortheentiresetofcomponents.Onlyundertheseconditionscanyouachieveoptimalyields. Top ProductsforinvitrotranscriptionInvitrogenoffersacompletelineofproductsforinvitrotranscription.TheMAXIscriptKitisidealformakingradio-andnonisotopically-labeledRNAprobesforuseinhybridizations.TheprobesgeneratedbytheInvitrogen™Strip-EZ™RNAProbeSynthesisandRemovalKitsarereadilystrippedfromNorthernblots,enablingmanyroundsofhybridizationwithoutdamagingnucleicacidboundtotheblot.TheMEGAscriptfamilyofkitsuseInvitrogen'shigh-yieldpatentedtechnologytosynthesizeRNAforapplicationswherelargemassamountsarerequired.LargeamountsofcappedRNAtranscriptscanbesynthesizedwiththeInvitrogen™ mMESSAGEmMACHINE™Kitusingthesamehigh-yieldpatentedtechnology.Ifdesired,theInvitrogen™ Poly(A)TailingKitcanbeusedtoaddapoly(A)tailtocappedRNAtranscriptssynthesizedwiththemMESSAGEmMACHINEKit.TheInvitrogen™ MessageAmp™aRNAKitisacompletekitforaRNAamplificationbasedonthepatentedEberwinemethod.IncorporatingMEGAscripthigh-yieldtranscriptiontechnology,thiskitincludesallnecessaryreagentsforfirst-strandcDNAsynthesis,RNaseHdigestion,second-strandsynthesis,cDNApurification,invitrotranscriptionandaRNApurification.Top ReferencesLingM-L,RismanSS,KlementJF,McGrawN,McAllisterWT.AbortiveinitiationbybacteriophageT3andT7polymerasesunderconditionsoflimitingsubstrate.Nucl.AcidsRes.(1989)17:1605-1618.TopAM1308,AM1310,AM1312,AM1314,AM1316,AM1320,AM1322,AM1324,AM1326,AM1330,AM1334,AM1326,AM1330,AM1334,AM1338,AM1340,AM1344,AM1348,AM1350,AM1354,AM1750×MinimalSequenceRequirementsThe+1base(inbold)isthefirstbaseincorporatedintoRNAduringtranscription.Theunderlineshows theminimumpromotersequenceneededforefficienttranscription.Close Signin Don'thaveanaccount? 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