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Optimized,completesystemforproductionoffull-lengthCDNAfromlowamountsoftotalRNA.
Synthesizefull-lengthcDNA(>15kb)- Generatefirst-strandcDNAfrompicogramamountsoftotalRNA
- Selectolido(dT)orrandomprimers-bothincluded
Applications
- First-strandcDNAsynthesisforsubsequentPCRorreal-timePCR.
- RT-PCRvalidationofgeneexpressiondataobtainedfrommicroarrayexperiments.
- RT-PCRvalidationandquantificationofgenesilencingbyRNAinterference.
TheMMLVReverseTranscriptase1st-StrandcDNASynthesisKitproducesfull-lengthfirst-strandcDNAfromtotalcellularRNApreparationsorpurifiedpoly(A)RNA.
ThiscompletekitincludesthesameMMLVHighPerformanceReverseTransciptasethatisavailableasaseparatecomponent(Cat.No.RT80125K)andmaintainsallthesamehighperformancecharacteristicsasthatstandaloneenzyme.TohelpsimplifyyourcDNAsynthesis,thiskitcontainsallthenecessarycomponentstomake1ststrandcDNA.
Benefits
- Synthesizefull-lengthcDNAfromRNAtemplateslongerthan15kb.
- MMLVHPRTdemonstratessignificantlyhigheractivitythancompetitivereversetranscriptaseenzymes.
- ReactionBuffer,optimizedforproducingfull-lengthcDNA,isincludedwithboththeMMLVHPRTandtheMMLVReverseTranscriptase1st-StrandcDNASynthesisKit.
- Thekitincludesbothanoligo(dT)andrandomnonamerprimers.
- First-strandcDNAcanbemadefrompicogramamountsofinputtotalRNA.
- ThekitincludesapotentRNaseInhibitortoprotecttheintegrityoftemplateRNA.
Figure1.MMLVHPRTproducesfull-lengthcDNAfrommRNAlongerthan15kb.TotalRNAisolatedfromHeLacellswasreversetranscribedandthecDNAwasamplifiedbyPCR.Detectionofthe1.3-kbPCRampliconfromnearthe5´endofthemRNAdemonstratesfull-lengthreversetranscriptionofHERC1mRNA(A).AgarosegelanalysisofthePCRproductsshowsthe1.3-kbampliconfromthe5´endofthemRNA(B).LaneM,100bpDNAladder;lane1,no-RTcontrolreaction;lane2,PCRproductfromcDNAsynthesizedbyEpicentre'sMMLVHPRT.
| TargetTranscript(Size) | 3´/5´Ratios | ||
| EpicentreMMLVHighPerformanceReverseTranscriptase | CompetitorI(RNaseH-MutantofMMLVRT) | CompetitorP(MMLVRT) | |
| ACTB(1,792b) | 0.9 | 1.7 | 1.2 |
| GUSB(2,162b) | 1.0 | 6.1 | 2.5 |
| TFRC(5,010b) | 5.5 | 12.1 | 11.3 |
Table1.3´/5´ratioanalysisofcDNAproducedbydifferentreversetranscriptaseenzymes.TotalcellularRNAfromHeLacellswasconvertedtocDNAusingthethreereversetranscriptaseenzymesindicatedinthetable.A3´/5´ratioequalto1.0meansthatequalamountsofPCRproductsareobtainedfromboththe3´and5´endofthecDNAandthereforeisagoodindicationthatthereversetranscriptasehasproducedafull-lengthcDNAcopyofthemRNA.
2A.
Figure2.cDNAproducedbyEpicentre'sMMLVHighPerformanceReverseTranscriptaseyieldsasignificantlyimproved3´/5´ratiothancompetitivereversetranscriptases.Theapproximately2160-baseHeLaβ-glucuronidasemRNA(GUSB)wasreversetranscribedintocDNAusingEpicentre'sMMLVHighPerfomanceReverseTranscriptaseandtwocompetitivereversetranscriptaseenzymes.PCRprimerpairstothe3´-endand5´-endofGUSBcDNAweresynthesizedandqPCR(SYBR®GreenIdyedetection)wasperformedusingeachprimerpairandtheGUSBcDNAsastemplates.The3´/5´ratiowascalculatedforeachasdescribedinthetext.(A).PCRampliconsfromthe3´endand5´endoftheGUSBcDNA.(B).qPCRquantificationgraphsfordetectingthe3´ampliconand5´ampliconofGUSBcDNAproducedbyEpicentre'sMMLVHighPerformanceReverseTranscriptaseKitandtwoothercommerciallyavailablereversetranscriptaseenzymes.
| 5'-amplicon | |
| 3'-amplicon |
2B.
EpicentreMMLV1st-StrandcDNASynthesisKit
CompanyIRNaseH-minusMMLVRT
CompanyPMMLVRT
