Poly(A) Tailing Kit | Thermo Fisher Scientific - US

The Poly(A) Tailing Kit uses E. coli Poly(A) Polymerase I (E-PAP) to polyadenylate the 3' termini of in vitro transcribed RNA. Polyadenylation plays an ... PopularApplications&TechniquesShopAllProductsServicesSupport Signin QuickOrder     Search ThermoFisherScientific SearchAll Search Home›TechnicalReferenceLibrary›RNATechnicalResourcesfromAmbion›LargeScaleTranscription›TechNotes›Poly(A)TailingKitQuickandEasyAdditionofPoly(A)TailstoInVitroTranscribedRNASeeNavigation‹TechNotesCappedRNAthatGivesHigherProteinYieldsmMESSAGEmMACHINET7Ultra ›HighYieldTranscriptionforEveryApplication|FromtheRecognizedLeaderinHighYieldTranscription ›Poly(A)TailingKit ›RapidlySynthesizeLargeAmountsofCappedRNA ›RNAiinNon-MammalianCulturedCells ›SynthesisandBiologicalApplicationsofCapAnalogswithSuperiorTranslationalProperties ›YieldIsn'tEverythingInAHighYieldTranscriptionKit! ›NEW!Poly(A)TailingKitAddsapoly(A)tailofatleast150nucleotidestothe3'terminiofRNAEnhancestranslationalefficiencyinvivoAdjustablereactiontocontrolfortaillengthOptimizedforusewithRNAsynthesizedusingthemMESSAGEmMACHINE™KitThePoly(A)TailingKitusesE.coliPoly(A)PolymeraseI(E-PAP)topolyadenylatethe3'terminiofinvitrotranscribedRNA.PolyadenylationplaysanimportantroleinthestabilizationofRNAineukaryotesandenhancestheefficiencyoftranslationinitiation.AM1340,AM1344,AM1348,AM1350 EnhanceTranslationUpto10X E-PAPisa53kDterminaladenylyltransferasewithhighspecificityforATPandRNAsubstrates.E-PAP'snormalfunctionistopolyadenylatemRNAsthatthenbecometargetsfordegradationbyintracellularexonucleases.However,reportshaveshownthatpoly(A)tailingofinvitrotranscriptsincreasemRNAstabilitywheninjectedintoXenopusoocytes(1).InthePoly(A)TailingKit,AmbionhasoptimizedtheE-PAPreactionsothatmRNAsareefficientlytailedwithatleast150adenines.Figure1showsvarioussizedtranscriptsbeforeandafteraPoly(A)TailingKitreaction.Theincreasedtranscriptsizeinthe"after"lanesisaresultofpolyadenylation.TheadditionaladenineresiduesconferstabilitytothemRNAresultinginincreasedtranslationalefficiencyofinvitrosynthesizedcappedRNAinmicroinjectionandtransfectionexperiments(2,3,4). Figure1.TailingofVariablySizedTranscripts.Transcriptsof0.5kb,1.5kband4kbweregeneratedfrommMESSAGEmMACHINE™reactionsand10µgofeachweretailedwith2unitsofE-PAPfor1hourat37°C.0.5µlofeachreaction,pre-andpost-tailing,wererunona1%denaturingformaldehyde-agarosegelin1XMOPSbuffer,aspertheprotocol.Laneswerevisualizedbytheadditionof50µg/mlethidiumbromideintothegelloadingbufferandviewedonaUVlightbox. Transcriptswithpoly(A)tailsshowincreasedproteinsynthesisby5-10foldcomparedtoun-tailedmessageswhentransientlytransfectedintoeukaryoticcells.HeLacellsweretransfectedwith1µgofcappedonlyorcappedandpolyadenylatedluciferasetranscriptsatvarioustimepoints.Thecappedandtailedtranscriptexpressed5.2-foldmoreproteinoverthecappedonlytranscriptasdeterminedbyluciferaseassay(Figure2). Figure2.EffectofPoly(A)TailonTranslation.HalfofaluciferasetranscriptpreparationsynthesizedwiththemMESSAGEmMACHINEKit™waspolyadenylatedusingthePoly(A)TailingKit.AmicrogramofeitherpolyadenylatedandunpolyadenylatedtranscriptwastransfectedintoHeLacells.Luciferaseproteinwasmeasuredinthecellsovervarioustimepointsposttransfection. Quick,ControlledPolyadenylationofinVitroTranscripts Whenshorterpoly(A)tailsarerequired,theamountofE-PAPenzymecanbereducedtoproduceacorrespondingdecreaseintaillength.InFigure3,approximately10µgofa188basehumanß-actintranscriptwastailedat37ÜCfor1hourwithvaryingamountsofE-PAP.Theproductswererunona2.5%denaturingagarosegelandstainedwithethidiumbromide.Figure3.TitrationofE-PAPintoaTailingReaction.Humanß-actin188basecontroltranscript(10µg/rxn)fromamMESSAGEmMACHINE™reactionwastailedwithdecreasingamountsofE-PAPfor1hourat37°C.0.5µlofeachreactionwasrunona2.5%denaturingformaldehyde-agarosegelin1XMOPSbuffer,aspertheprotocol.Laneswerevisualizedbytheadditionof50µg/mlethidiumbromideintothegelloadingbufferandviewedonaUVlightbox. ThePoly(A)Tailingreactionissimpleanddoesnotrequirethetranscripttobepurified.AfterinvitrotranscriptionusingthemMESSAGEmMACHINE™Kit,theE-PAPenzymeandoptimizedbufferareaddeddirectlytothetranscriptionreactionandasecondincubationisperformedat37°Cfor1hour.Thetranscriptisnowcapped,tailed,andreadyforuse.ThePoly(A)TailingKitcontainsE-PAPenzyme,buffer,ATPandtheothernecessaryreagents.ThePoly(A)TailingKitisoptimizedforusewithAmbion'smMESSAGEmMACHINE™HighYieldCappedRNATranscriptionKit. References Belasco,J,Brawerman,G(1993)ControlofmessengerRNAstability.AcademicPress,SanDiego,CA.DrummondDR,ArmstrongJ,ColmanA(1985)StabilityandmovementofmRNAsandtheirencodedproteinsinXenopusoocytes.JCellBiol100(4):1148-1156.GaliliG,KawataEE,SmithLD,LarkinsBA(1988)Roleofthe3'-poly(A)sequenceintranslationalregulationofmRNAsinXenopuslaevisoocytes.JBiolChem.263(12):5764-70.WakiyamaM,FutamiT,MiuraK(1997)Poly(A)dependenttranslationinrabbitreticulocutelysate.Biochemie79(12):781-785.  Signin Don'thaveanaccount? 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