EpiScript™RnaseH^-ReverseTranscriptase(EpiScriptRT),analternativetoSuperScript®IIReverseTranscriptase,isarecombinantMMLVreversetranscriptasewithgreatlyreducedRNaseHactivity.Itishighlyefficientatproducingfull-lengthcDNAfromlongRNAtemplates.EpiScriptRTiscapableofproducingcDNAfromaslittleas50pgoftotalRNAforreal-timeRT-PCR(qRT-PCR)analysisandotherapplications.

Applications

• First-strandcDNAsynthesisforsubsequentPCRorreal-timePCR.

Benefits

• RecombinantMMLVreversetranscriptasewithgreatlyreducedRNaseHactivity
• Activeattemperaturesupto55°C
• Highlyefficientatproducingfull-lengthcDNAfromaslittleas50pgoftotalRNA
• BestvalueinanRNaseH-ReverseTranscriptase

Storage:Storeonlyat-20°Cinafreezerwithoutadefrostcycle.

StorageBuffer:EpiScriptRTissuppliedina50%glycerolsolutioncontaining50mMTris-HCl(pH7.5),100mMsodiumchloride,1mMDTT,0.1mMEDTA,and0.1%Triton®X-100.

UnitDefinition:OneunitofEpiScriptRTcatalyzestheincorporationof1nmolofdTTPintoacid-insolublematerialin10minutesat37°Cusingsaturatingamountsofoligo(dT)-primedpoly(rA)astemplate.

ContaminatingActivityAssays:EpiScriptRTisfreeofdetectableexonuclease,endonuclease,andRNaseactivities.

Figure1.EpiScript™ReverseTranscriptaseperformedequallyorbetterthancomparablereversetranscriptasesfromothervendors.First-strandsynthesisreactionswereassembledaccordingtomanufacturer´sspecifications.InputRNAwas1µgofJurkattotalRNA(Ambion®).Reactionswereprimedusing50ngofpoly-T_(16-18)DNA.2ndstrandqPCRwasperformedusingBio-RadiQSYBRmastermixandgene-specificprimersthatyielded250-350bpamplicons.Reactionswererepeated4-fold.PGDF-R(PlateletDerivedGrowthFactorReceptor),TNF(TumorNecrosisFactor),IL-1b(Interleukin-1beta),IL-2(Interleukin2).ImagecourtesyofMatthewKellinger,Illumina®Inc.

Figure2(clicktoenlarge).EpiScript™ReverseTranscriptaseproducessimiliartranscriptcoverageindependentoftranscriptlength.EpiScriptwasusedtoprimefirststrandcDNAeitherfromtotalRNAusingoligo-dT(leftpanel)orpolyA+selectedRNAwithrandomhexamers(rightpanel).ThecDNAwasconvertedintoIllumina®-compatIBLelibrariesandsequenced.Thereadswerealignedtotranscriptsbaseduponvariouslengthclassesandreaddensityplottedrelativetothepercentdistancefromthe5´endofthetranscripts;0%referstothe5´endand100%isthe3´end.Asexpected,oligo-dTprimingresultsinamorepronounced3´biasthanrandompriming.

Figure3.UseofEpiScript®ReverseTranscriptaseforfirststrandcDNAresultsindetectionofsimilartranscriptcategoriesindependentofinputamount.EpiScriptRTwasusedtorandomprime5ngor50pgofhumantotalRNAandthecDNAwasconvertedintolibrariesforIllumina®sequencing.ReadswerealignedusingTophatandannotatedwithCufflinksandthepercentageofeachmajorcategoryisvisualizedasapiechart.Equivalentresultsareobservedforboth5ngand50pgofinputRNA.