NewProducts BeSMART™AboutFirst-StrandcDNASynthesis SMARTScribe™ReverseTranscriptase—theRTofchoiceforallofourSMARTKits •Synthesizelong,full-lengthcDNA •Amplifyraretranscripts •MaintainplexityoftheoriginalRNA •Robustsystemwithconsistentperformance Reliablereversetranscriptionrequiresaqualityreversetranscriptase(RT)thatcanefficientlygeneratefull-length,first-strandcDNAfromavarietyofRNAtranscripts.SMARTScribeReverseTranscriptaseisahigh-performanceenzymethatallowsunbiasedcDNAsynthesisandlibraryconstructionfromanyRNAtranscript.SMARTScribeRTisamodifiedMoloneyMurineLeukemiaVirusReverseTranscriptasethatgenerateslong,full-lengthcDNAandamplifiesraretranscripts,whilepreservingtherelativetranscriptproportionsoftheoriginalRNAsample.ItistheidealRTtousewithallofourSMARTKits
(1).
1 2
3 4 kb –10.0 –8.0 –6.0 –4.0–3.0 –2.0–1.5 –1.0 Figure1.SMARTScribeRTgenerateslong,first-strandcDNA.SMARTMMLVRT(Lanes1&2)andSMARTScribeRT(Lanes3&4)wereeachusedtosynthesizefirst-strandcDNAusingapolyA+RNAladderastemplate.Thesampleswereanalyzedbydenaturingagarosegelelectrophoresis.SMARTScribewasabletogeneratelargeamountsofsingle-strandedcDNAfromRNAofallsizes,includingthe10kbtranscripts. A mRNA5'RT 5'PCR3'PCRprimerprimer AAAA–3'TTTT–5'
B M12345678910kb1.0– 0.5– Figure2.SMARTScribeRTsynthesizeslong,full-lengthcDNA.SMARTScribewasusedtoreversetranscribefirst-strandcDNAfromHumanUniversalReferenceTotalRNAusingoligo(dT)18primers(PanelA).Theresultingsingle-strandedcDNAwasthenanalyzedbyPCRusingprimersthatgenerated200–800bpampliconsfromthe5'endsof10genes(PanelB,Lanes1–10).SeeTableIforinformationonthegenesanalyzedandthesizeoftheamplicongeneratedfromeach;thelanenumberscorrespondtothesamplenumberinthetable.TheessfulgenerationofeachampliconindicatesthatSMARTScribeRTwasabletosynthesizesingle-strandedcDNAtranscriptsaslongas14.7kb(fromLRP2mRNA;Lane10).LaneM:100bpDNAsizemarker. TableI:GenesAnalyzedbyRT-PCRandtheMinimumLengthsofthecDNATranscriptsGenerated SampleessionNo.GeneName AmpliconSize(bp) MinimumLengthofcDNATranscript (bp)
1 NM_002223Humaninositol1,4,5-triphosphatereceptor,type2(ITPR2) 599 11,607
2 NM_000267Humanneurofibromin1(NF1) 554 11,739
3 NM_001408Humancadherin,EGFLAGseven-passG-typereceptor3(CELSR3) 572 10,057
4 NM_057164Humancollagen,typeVI,alpha3(COL6A3) 811 9,122
5 NM_015092HumanPI-3-kinase-relatedkinaseSMG-1(SMG1) 224 13,007
6 NM_000426Humanlaminin,alpha2(LAMA2) 597 8,598
7 NM_000059Humanbreastcancer2,earlyonset(BRCA2) 792 10,158
8 NM_004010Humandystrophin(DMD) 644 13,166
9 NM_002332Humanlowdensitylipoprotein-relatedprotein1(LRP1) 598 13,432 10 NM_004525Humanlowdensitylipoprotein-relatedprotein2(LRP2) 563 14,652 ClontechLaboratories,Inc.• ClontechniquesJanuary200921 NewProducts BeSMART™AboutFirst-StrandcDNASynthesis…continued NumberofRNAcopies NoRNA
M 105 104 103 102 10 control A12345678 300bp 350bp B12345678 Figure3.SMARTScribeRTexhibitsexceptionalsensitivity.RT-PCRassayswereperformedusingSMARTScribeRTandsyntheticRNA.ThesyntheticRNAwasseriallydiluted10Xtoobtain105–10copiespersample;dilutedtemplatewasthenspikedinto50ngoftotalRNAfromHeLacellstoplexity.First-strandcDNAwassynthesizedwithSMARTScribeRT,thenamplifiedwithAdvantage®2DNAPolymeraseMixin36cyclesofPCRusingprimersspecificfora350bpamplicon.ThePCRreactionswerethenanalyzedbyagarosegelelectrophoresis.SMARTScribewasabletogeneratesingle-strandedcDNAfromasfewas10copiesofsyntheticRNA.LaneM:100bpDNAsizemarker. GenerateLong, Full-LengthcDNA SMARTScribeRTcontainsmodificationsthatallowoptimalreversetranscriptionofvirtuallyanyRNA,includingrareandlongtranscripts(Figure1).Inaddition,theenzymeissubjecttothesameproprietarypurificationprocessasSMARTMMLVRT.ThisensuresthatSMARTScribepreparationsareexceptionallypure,withallcontaminatingnucleasesremoved. SMARTScribe’sabilitytosynthesizelong,full-lengthcDNAwasdemonstratedinreversetranscriptionPCRassays(RT-PCR;Figure2)inwhichSMARTScribeRTwasusedtogeneratefirst-strandcDNAfromHumanUniversalReferenceTotalRNA.Theresultingsingle-strandedcDNAwasthenanalyzedviaPCRreactionsthatgeneratedshort(200–800bp)ampliconsfromthe5'regionofeachofthegeneslistedinTableI(Figure2,PanelsAandB).TheessfulgenerationoftheseampliconsdemonstratesthatSMARTScribeRTwasabletosynthesizesingle-strandedcDNAtranscriptsofupto14.7kb(TableI;Figure2,PanelB,Lane10).Theabilitytoproducelong,high-qualitycDNAmakesSMARTScribeRTtheenzymeofchoiceforallapplicationsrequiringlong,full-length,first-strandcDNA. AmplifyRareorLow CopyTranscripts SMARTScribeRTexhibitsexceptionalsensitivity,whichresultsinmaximumamountsoffirst-strandcDNAregardlessoftemplatesizeorabundance.Asaresult,rareorpreciousRNAsamplescanbepreserved.ThiswasdemonstratedinRTPCRassays,whereSMARTScribeRTwasusedtogeneratefirst-strandcDNAfromeithersyntheticRNAtemplate(Figure3)orHumanUniversalReferenceTotalRNA(Figure4,PanelA).Theresultingsingle-strandedtranscriptswereamplifiedbyPCRandvisualizedonagarosegels.SMARTScribewasabletosynthesizesinglestrandedcDNAfromasfewas10copiesofsyntheticRNA(Figure3),andaslittleas0.1pgoftotalRNA(Figure4,PanelA,Lane8). MoreSensitivethan theCompetition paredthesensitivityofSMARTScribeRTwiththatofpetitorenzymesintheRT-PCRassayinFigure4.Inthisassay,eachenzymewasusedtoreversetranscribeHumanUniversalReferenceTotalRNAthathadbeenseriallydiluted 300bp C12345678 300bp Figure4.SMARTScribeRTismoresensitivethanpetition.HumanUniversalTotalRNAwasseriallydiluted10X(from1μgto0.1pg),thenreversetranscribedusingeitherSMARTScribeRT(PanelA),CompetitorP(PanelB)orCompetitorQ(PanelC).EachRTreactionwasperformedordingtotheenzymemanufacturer’sspecifications.OnemicroliterofeachRTreactionwasthenusedinPCRreactionswithprimersspecificfora300bpportionofthe5'endoftheβ-actingene.Thesampleswereanalyzedbyagarosegelelectrophoresis.TheamountoftemplateRNAusedineachreactionisasfollows:Lane1:1μg.Lane2:100ng.Lane3:10ng.Lane4:1ng.Lane5:100pg.Lane6:10pg.Lane7:1pg.Lane8:0.1pg.SMARTScribeRTexhibitedsuperiorsensitivityatallofthetemplateconcentrationstested. 10Xfrom1μgto0.1pg.TheresultingsinglestrandedcDNAwasthenusedinPCRassaystogeneratea300bpampliconfromthe5'endofβ-actincDNA.Thesampleswereanalyzedbyagarosegelelectrophoresispared.SMARTScribeRT(Figure4,PanelA)exhibitedsuperiorsensitivitydownto0.1pgtotalRNA—thelowestRNAconcentrationused. 22ClontechLaboratories,Inc.• ClontechniquesJanuary2009 NewProducts BeSMART™AboutFirst-StrandcDNASynthesis…continued Perfectforall SMARTApplicationsSMARTScribeRTistheidealenzymeforthemostdemandingSMARTapplications,andhasbeenformulatedspecificallyforusewithallofourSMARTKits.FortheassayinFigure5,SMARTScribewasusedwithourSMARTRACEcDNAAmplificationKittoamplifythe5'regionofthetransferrinreceptor(TFR)genefromHumanPlacentaTotalRNA(Figure5).ThiskitcontainsaspeciallydesignedSMARTOligothatincreasesthelikelihoodofcloningentiregenesequencesorupstreamregulatoryregions.Together,SMARTScribeRTandtheSMARTRACEcDNAAmplificationKitwereabletoamplifyTFRcDNAfromaslittleas0.5ngtotalRNA(Figure5,Lane3). WhichClontechRT ShouldIUse?
ClontechnowofferstwoRTs—SMARTMMLVandSMARTScribe: •Botharehighlypurifiedenzymeswithallcontaminatingnucleasesremoved. •SMARTScribeRThasbeenengineeredtoprovidethebestperformancewhenamplifyingrareorlongtranscripts,orinapplicationswhereitisimportanttofaithfullymaintaintheproportionsoftheoriginalRNAtranscripts.Forthisreason,wemendtheuseofSMARTScribeRTwithmoredemandingSMARTKitapplications.
M 1
2 3
4 5
6 kb 3–2– Figure5.SMARTScribeisspeciallyformulatedplementallofourSMARTKits.SMARTScribewasusedinconjunctionwithourSMARTRACEcDNAAmplificationKittoamplifytransferrinreceptor(TFR,2.6kb)cDNAfromHumanPlacentaTotalRNA.TemplateRNAwasseriallydiluted2Xtoobtain0.25ng–2ngtemplatepersample(Lanes2–5,respectively).Controlsamplescontainingnotemplate(Lane1)and50ngtemplate(Lane6)werealsoused.Together,SMARTScribeandtheSMARTRACEcDNAAmplificationKitwereabletoamplifyTFRcDNAfromaslittleas0.5ngtotalRNA(Lane3).LaneM:1kbladderDNAsizemarker. •SMARTMMLVRThasbeenusedwithourSMARTKitsbymanyscientistswhohavereportedexcellentresults;itisanexcellentenzymeforlessdemandingSMARTapplications.SMARTMMLVRThasalsobeenshowntoprovidehigheryieldsforquantitativereversetranscriptionPCR(qRT-PCR)thandootherenzymes. Ingeneral,whenperformanceiscritical,wemendusingSMARTScribeRTwithourSMARTKits,andSMARTMMLVRTforqRT-PCR.However,thisisnotadefinitivemendation,astheenzymesmayperformdifferentlyundertheconditionsrequiredforaparticularapplication. SMARTScribeRTistheidealhighperformancereversetranscriptaseforunbiasedcDNAsynthesis,mRNAamplification,orlibraryconstructionfromanyRNAtranscript.Tryitbyitself,orastheplementtoanyofourSMARTKits. Product Size Cat.No. SMARTScribeReverseTranscriptase NEW!
40rxns 639536 100rxns 639537 400rxns 639538 SMARTMMLVReverseTranscriptase8,000units63952320,000units639524 Components •SMARTScribe™ReverseTranscriptase•20mMDTT•5XFirst-StrandBuffer RelatedProducts •SMART™RACEcDNAAmplificationKit(Cat.No.634914) •SMART™PCRcDNASynthesisKit(Cat.No.634902) •SuperSMART™PCRcDNASynthesisKit(Cat.No.635000) •SMART™cDNALibraryConstructionKit(Cat.No.634901) •SMART™mRNAAmplificationKit(Cat.No.635001) •Advantage®2DNAPolymerase(CatNos.639201,639202,639206&639207) •HumanUniversalReferenceTotalRNA(Cat.No.636538) •HumanPlacentaTotalRNA(Cat.No.636527) References
1.Chenchick,A.etal.(1998)GenerationandUseofHigh-QualitycDNAfromSmallAmountsofTotalRNAbySMART™PCR.InGeneCloningandAnalysisbyRT-PCR.Eds.Siebert,
P.&Larrick,
J.(BiotechniquesBooks,MA)Ch22. ClontechLaboratories,Inc.• ClontechniquesJanuary200923