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
ORDERINFORMATION
MMLVReverseTranscriptase,10XReactionBuffer,RiboGuard™RNaseInhibitor,dNTPPreMix,DTT,Oligo(dT)21Primer,Random9-merPrimers,RNase-freeWater.ebiomall.com
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高等物般具105种左右同基,定间阶段单细胞或体,都尽15%左右基表达,产约15000种同mRNAcDNA文库通RNA反转录由mRNA发cDNA克隆,其复杂程度要比直接基组克隆简单.
cDNA文库显然比基因组DNA文库小得多，能够比较容易从中筛选克隆得到细胞特异表达的基因。但对真核细胞来说，从基因组DNA文库获得的基因与从cDNA文库获得的不同，基因组DNA文库所含的是带有内含子和外显子的基因组基因，而从cDNA文库中获得的是已经过剪接、去除了内含子的cDNA。
真核生物基因组DNA十分庞大,其复杂程度是蛋白质和mRNA的100倍左右,而且含有大量的重复序列. 采用电泳分离和杂交的方法,都难以直接分离到目的基因.这是从染色体DNA为出发材料直接克隆目的基因的一个主要困难。
高等生物一般具有10^5种左右不同的基因,但在一定时间阶段的单个细胞或个体中,都仅有15%左右的基因得以表达,产生约15000种不同的mRNA分子.可见,由mRNA出发的cDNA克隆,其复杂程度要比直接从基因组克隆简单得多。
cDNA文库在研究具体某类特定细胞中基因组的表达状态及表达基因的功能鉴定方便具有特殊的优势，从而使它在个体发育、细胞分化、细胞周期调控、细胞衰老和死亡调控等生命现象的研究中具有更为广泛的应用价值，是研究工作中最常使用到的基因文库。
基因组文库是一个比较笼统的概念。这个文库可以指某种真核生物的基因组，也可以指某种原核生物的基因组。实质是将某种生物的全部遗传信息贮存在一个受体菌群体中，做为目的基因的来源。
