RT-PCR Product Listing R7647 O4387 M1302 R9376 Page AMPD1 J3520 HSRT100 HSRT20 STR1 Product Description A4464 Catalog Number • • • • Features Andbenefits up To14.1Kb

RT-PCR from Avian MyeloblastosisVirus Reverse Transcriptase eAMV™ RT-PCR Product Listing R7647 O4387 M1302 R9376 Page AMPD1 J3520 HSRT100 HSRT20 STR1 Product Description A4464 Catalog Number • • • • Features andBenefits up to14.1kb. through difficult secondarystructure andtranscribemRNAtemplates enhanced abilitytodetectlowabundancemessages,transcribe standard AMVorM-MLV reverse transcriptase.eAMVexhibitsan This exceptionallyrobust hasgreater AMVRT thermostabilitythan complementary toRNA,DNAoranRNA:DNAhybrid. thatsynthesizesaDNAstrand (AMV)RT Myeloblastosis Virus eAMV ReverseTranscriptase isanenhancedformofAvian AMV-RT orRNaseH-minusM-MLV RT Detects lowabundancemRNAbetterthanRNaseH-reduced than AMV-RT, M-MLV orRNaseH-minusM-MLV RT RT More efficient attranscribingthrough difficult secondarystructure Produces full-lengthcDNA,upto14.1kb,withhigheryields Produces firststrandcDNAready forPCRamplification RNA ReverseTranscribed upto14kbinLength 1234567891011 Deoxyribonuclease I,AmplificationGrade eAMV HSRT-PCRKit eAMV HSRT-PCRKit eAMV FirstStrandSynthesisKit Oligo(dT) M-MLV ReverseTranscriptase eAMV ReverseTranscriptase Random Nonamers AMV ReverseTranscriptase JumpStart REDHTRT-PCRKit 23 nhrd22 , Anchored 21 19 17 19 22 22 22 18 20 template andoligo(dT) into TCA-precipitablematerialin10minusingpolyadenylicacidas 2. Tosh, C.,etal.,One-tubeandone-buffer systemofRT-PCR 1. Brooks, E.M.,etal.,Secondarystructure inthe3’-UTRofEGF References Shipped inwetice 3. Dukas, K.P., etal.,Quantitationofchangesintheexpression of Concentration: Unit definition: Components: Storage: 10 ae1:452bpPCRproduct,880transcriptsize Lane 11: 349bpPCRproduct,1,060transcriptsize Lane 10: NegativeControl(NoRT) Lane 9: 300bpPCRproduct,14,150transcriptsize Lane 8: 396bpPCRproduct,12,890transcriptsize Lane 7: 499bpPCRproduct,10,980transcriptsize Lane 6: 608bpPCRproduct,9,970transcriptsize Lane 5: 708bpPCRproduct,6,875transcriptsize Lane 4: 796bpPCRproduct,5,760transcriptsize Lane 3: 908bpPCRproduct,2,180transcriptsize Lane 2: Marker Lane 1: Lanes 2-8areprimersetsforp619,Lane10is demonstrate theintegrityofcDNAupto14kb. tail resultinginthefollowingPCRproducts.Successfulamplifications Catalog NumberINSP1)chosenatdifferent distancesfromthepoly(A) using JumpStartREDTaq withPCRprimersets(RNAInspectorKit, using eAMVReverseTranscriptase. TheresultingcDNAwasamplified mRNA fromHEK293cellswasreversetranscribedtoalengthof14.1kb a.N.PoutDsrpinQuantity A4464 Product Description Cat. No. 3 isolates, amplification of1Dgenefoot-and-mouthdiseasevirusfield message diversitybyRT-PCR, and thechoiceofreverse transcriptasesaffect thedetectionof Analyt. Biochem. multiple genesbysimultaneouspolymerasechainreaction, Buffer foreAMVReverseTranscriptase –20 °C Acta Virol. eAMV ReverseTranscriptase eAMV ReverseTranscriptase 20 unitsper One unitincorporatesonenanomoleofTMP 215, 41, 12-18 66-72 (1993). 153-155 (1997). as aprimer μ Biotechniques l β -actin andLane11isGAPDH 19, 806-812 (1995). 1,000 units 500 units +

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RT-PCR 17 18 RT-PCR sigma-aldrich.com cDNA transcripts Going togreaterlengths—up14.1kb Synthesis Kit eAMV™ FirstStrand RT-PCR template andoligo(dT) into TCA-precipitable materialin10minusingpolyadenylicacidas including PCR. generated thatcanbeusedforvariousdownstream applications, thiskit,adependablecDNAis able reverse transcriptases.With difficult secondarystructure betterthanothercommercially avail- peratures, upto65°C,whichallowsittranscribethrough that are ofverylowabundance.Itismore stableatelevatedtem- Unit definition: poly(A) stable complementaryDNA(cDNA)from fragiletotalRNAor The eAMVFirstStrandSynthesisKitistheidealwaytogenerate + RNA. eAMVwilleffectively transcribemessages,evenones One unitincorporatesonenanomoleofTMP 12-18 as primer 1. Brooks E.M.,etal.,Secondarystructure inthe3’UTRofEGFand Reference Shipped indryice Random nonamers,100 Storage: Components: Anchored oligo(dT) Deoxynucleotide mix,50 10 PCR GradeWater, 1.5ml RNase Inhibitor, 1vial a.N.PoutDsrpinQuantity STR1 Product Description Cat. No. 3 sage diversitybyRT-PCR. the choiceofreverse transcriptasesaffect thedetectionofmes- Buffer foreAMVReverseTranscriptase, 1.5ml –20 °C eAMV FirstStrandSynthesisKit Sufficient for50reactions eAMV ReverseTranscriptase, 1,000units 23 , 100 μ μ l l μ Biotechniques l 19, 806-812 (1995). 1 kit RT-PCR ae 0 1 8.9kbAPC 6.8 kbPol Lanes 10,11: 5.3 kbTSC-2 Lanes 8,9: 3.5 kbPol Lanes 6,7: 1kbLadder 2kb Pol Lanes 4,5: Lanes 2,3: Lane 1: Different poly(A) H-minus enzymeswhentranscribinglongcDNA. eAMV offers superiorperformance inlengthandyieldoverM-MLV RNase templates andlowabundancemessages The bestchoiceforamplificationofdifficult eAMV™ HSRT-PCR Kits Greater Transcription LengthsthanM-MLV RT RNaseH-minus 01 011 10 9 8 7 6 5 4 3 2 1 11 10 9 8 7 6 5 4 3 2 1 • • • • Features andBenefits the versatilityofaone-steportwo-stepprotocol. enzymes intooneRT-PCR kitprovides aqualitysystemthatoffers sensitivity oftheamplifiedproduct. Thecombinationofthesetwo AccuTaq LADNAPCRPolymerasewhichincreases specificityand scriptase attemperatures upto65°C.ThiskitincludesJumpStart ondary structures andmakeseAMVthemostrobust reverse tran- up to14.1kb.Itsbroad rangeofthermalactivityhelpsdisruptsec- through difficult secondarystructure andgeneratecDNAtemplates Transcriptase isabletodetectlowabundancemessages,transcribe and reliable systemwithanumberofadvantages.eAMVReverse gene expression. TheeAMVRT-PCR Kitprovides afast,convenient Reverse Transcription PCR(RT-PCR) isapowerfultoolusedtostudy LA DNAPolymeraseforlongandaccurateamplification Increased sensitivity, specificityandyieldfrom JumpStartAccuTaq reverse transcriptases Transcribes through difficult secondarystructure betterthanother transcriptases is abletotranscribeRNAundetectableotherreverse Higher sensitivityfordetectinglowabundancemessages.eAMV generating cDNAupto14.1kb Greater transcriptionlengthsthanotherreverse transcriptases, eAMV RT + RNA wereusedasatemplateintwo-stepRT-PCR. Competitor M-MLV RT RNase H-minus Shipped indryice of HeLapoly(A) Duplicate RT reactionswereperformedforeachenzymeusing50 8-13), usingeAMV, RNaseH-reducedAMV, andRNaseH-minusM-MLV. RNA, (Lanes2-7)andhumanHPRT, amediumabundanceRNA(Lanes RT-PCR wasperformedonhumanphopholipaseA2,alowabundance Only eAMVDetectsLowAbundanceMessage Storage: Components: at anelevatedtemperaturenecessary. region withextensivesecondarystucture,makingreversetranscription secondary structure.TheprimerusedfortheRT reactionislocatedina RT-PCR wasperformedon1.7kbTMVtranscriptcontainingdifficult PCR GradeWater Ribonuclease Inhibitor 10 mMdNTPMix 10 ae -3 SameenzymeorderasLanes2-7 Lanes 8-13: RT-PCR usingRNaseH-minusM-MLV DNAmarker Lanes 6and7: RT-PCR usingRNaseH-reducedAMV Lanes 4and5: RT-PCR usingeAMVRT Lanes 2and3: Lane 1: together beforePCR.Two Random NonamersandAnchored Oligo(dT) JumpStart AccuTaq LADNAPolymerase a.N.PoutDsrpinQuantity HSRT20 Product Description Cat. No. HSRT100 3 Reaction Buffers More RobustActivityatElevatedTemperatures –20 °C o bnac mediumabundance low abundance + eAMV HSRT-PCRKit eAMV HSRT-PCRKit Sufficient for100reactions Sufficient for20reactions RNA andreactionsfromeachenzymewerepooled eAMV ReverseTranscriptase eAMV μ l ofcDNAwasusedforeachPCRreaction. S tan d 23 ar Primers d AMVRT 467 bp 706 bp 1 kit 1 kit μ g

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RT-PCR 19 20 RT-PCR sigma-aldrich.com ae ,9 5 9 1ng Lanes 5,9,15,19: 10ng Lanes 4,8,14,18: 100ng Lanes 3,7,13,17: 1000ng Lanes 2,6,12,16: PCRMarker Lanes 1,10,11,20: ing tosuppliers’protocols. 1000 ngto1ng.Reactionswereperformedwith RT-PCR wasperformedontotalRNA-HELAcellsseriallydilutedfrom JumpStart™ REDHTRT-PCR Kit RT-PCR secondary structure. detection oflowabundanceRNA,especiallythosewithdifficult scriptases. Theseuniquefeatures makeittheidealenzymefor processivity andthermalstabilitycompared tootherreverse tran- critical forcomparativegeneexpression studies.eAMVhassuperior The sensitivitytolowabundancemessageprovided byeAMVis Thermal Stability eAMV HasSuperiorPerformanceand • • • • Features andBenefits DNA Polymerase. ReverseTranscriptaseVirus (eAMV-RT™) andJumpStartREDTaq™ contains acombinationofSigma’s EnhancedAvian Myeloblastosis point detectionoftargetRNAinacomplexsamplematrix.Thekit throughput gel-basedRT-PCR applicationsandisoptimizedforend- profiling. TheJumpStartREDHTRT-PCR Kitisdesignedforhigh A novelhighthroughput solutionforgel-basedgeneexpression Maximum flexibilityandminimumoptimization Direct sampleloadingafterPCR Enhanced sensitivityandspecificity Greater transcriptionefficiency 1 op Comp. I, VIII Comp. E 2345678910 11 op eAMV Comp. Q 21 41 61 81 20 19 18 17 16 15 14 13 12 β actin primersaccord- Shipped inwetice eAMV RT 10 eAMV RT JumpStart REDTaq DNAPolymerase Storage: Random Nonomers Ribonuclease Inhibitor Oligo dTPrimers PCR GradeWater 10 mmDeoxynucleotideMix Magnesium Chloride Components: 10 a.N.PoutDsrpinQuantity J3520 Product Description Cat. No. 3 PCR Buffer withoutMgCl –20 °C 3 JumpStart REDHTRT-PCRKit Buffer eAMV ReverseTranscriptase 2 200 reactions 40 reactions RT-PCR DNase I,AmplificationGrade • • • Features andBenefits leading molecularbiologyproduct suppliers. demonstrates lowerlevelsofRNaseactivitythanthatseveral comparisons haveshownthatSigma’s AmplificationGradeDNaseI valuable RNAsamplespriortoreverse transcription.Laboratory residual Many commercial DNaseIformulationsare contaminatedwith in reverse transcription. denatures hairpinsintheRNA,soRNAcanbeuseddirectly heat inactivationinthepresence ofStopSolution.Heatingalso the ReactionBuffer provided. TheDNaseIistheninactivatedby preparations ina15minutedigestionatroom temperature using DNA intooligo-andmononucleotides.isremoved from RNA of contaminatingDNA.DNaseIdigestsdoubleandsingle-stranded should berunwithoutreverse transcriptasetocheckforamplification should betreated withDNaseIbefore RT-PCR, andcontrol reactions Because PCRcanamplifyevenasinglemoleculeofDNA,RNAsamples Includes optimized10 Lowest RNaseactivityavailable applications suchasRT-PCR Ideal foreliminatingDNAfrom RNApreparations priortosensitive RNases. ThisRNasecontaminationcandestroy ordegrade 3 Reaction Buffer andStopSolution Shipped inwetice C Components: C analyzed ona1%agarosegel. incubated with1unitof assay wascompleted:1 For SigmaDNaseI,andforeachcompetitor’s DNaseI,thefollowing Storage: Unit definition: amplification ofcontaminatingDNA. reactions shouldberunwithoutreversetranscriptasetocheckfor Note: To determinetheeffectiveness ofDNaseItreatment,controlPCR 37 °Cfor1hour). Stop Solution,1ml 10 to oligonucleotidesin10minat37°C i u a.N.PoutDsrpinQuantity AMPD1 Product Description Cat. No. 3 = unincubatedcontrol(RNAinbuffer withoutDNase,keptonice). = incubatedcontrol(RNAinbuffer withoutDNase,incubatedat Reaction buffer, 1ml –20 °C

C Sigma AmplificationGradeDNaseI Deoxyribonuclease I,1kit Amplification Grade u DNase I,1,000units Has theLowestRNaseActivity C One unitcompletelydigests1 i Sigma μ g ofa1.9kb the respectiveDNaseIat37°Cfor1hourand

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RT-PCR 21 22 RT-PCR sigma-aldrich.com Random Nonamers Oligo(dT) Recombinant, expressedinEscherichiacoli M-MLV ReverseTranscriptase from Avian MyeloblastosisVirus AMV ReverseTranscriptase RT-PCR Technical andHSRT20). BulletinforCatalogNumbersHSRT100 0.1 mlissufficient for100RT-PCR reactions (asdescribedinthe precipitable materialin10minat37°C Shipped indryice template andoligo(dT) TCA-precipitable materialin10minusingpolyadenylicacidas dard oligo(dT)primerswhengeneratingcDNAfrom poly(A) Technical and HSRT20). BulletinforCatalogNumbersHSRT100 0.1 mlissufficient for100RT-PCR reactions (asdescribedin the synthesis, cDNAlibraryconstruction, RT-PCR andotherapplications. (9-mers) whichmaybeusedasuniversalprimersinfirststrandcDNA Random Nonamersare randomsequencesofninedeoxyribonucleotides anchored oligo(dT) such thatthere are nolongregions ofunusablesequence.Thus, The Anchored Oligo(dT) Concentration: Unit definition: Components: on aplasmid. The enzymeispurifiedfrom strand cDNAsynthesisforuseinRT-PCR reactions. M-MLV isusedforthepreparation ofcDNAlibrariesorforfirst hybrid (usingaprimer)tosynthesizecomplementaryDNAstrand. DNA polymerasethatusessingle-strandedRNA,oranRNA-DNA M-MLV ReverseTranscriptase (MoloneyMurineLeukemiaVirus) isa Storage: Concentration: Unit definition: cDNAs from mRNAforeventualcloningoruseasprobes. tion oftranscription.Thisenzymeiscommonlyusedtomake plate andadivalentcation,eitherMgorMn,are required forinitia- templates (cDNA).ADNAprimercomplementarytotheRNAtem- AMV ReverseTranscriptase synthesizesDNAcomplementarytoRNA 10 that theoligo(dT) one G,CorAresidue (theanchor)atthe3’end.Thisanchorensures 3 M-MLV ReverseTranscriptase Buffer withDTT –70 °C 23 M-MLV ReverseTranscriptase 23 , Anchored 23 200 unitsper 10,000-100,000 unitsperml One unitincorporatesonenmolofTTPinto primer bindsatthebeginningofmessage One unitincorporates1nmolofTTPintoacid- primers mayprovide anadvantageoverstan- 12-18 23 Primers have23thymidineresidues and as aprimer E. coli μ expressing the l pol gene ofM-MLV + RNA. Shipped inwetice Shipped inwetice Storage: 1. Breathnach, R.,etal., References Storage: Concentration: Storage: Concentration: 1. Howland, P., etal.,Positive-andnegative-actingpromoter References Shipped inwetice 3. Verma, I.M., S.M.,etal., 2. Tilghman, 2. Gerard, G.F. andD’Alessio,J.M.,Buwell,M.,Enzymesof 6. Berger, S.L.,etal., 5. Okayama,H.andBerg,P., 4. Sambrook,J.andRussell,D.W., 3. Gerard, G.F., etal.,Influenceonstabilityin 8. Champoux,J.J.,etal., 7. Leis,J.P., etal., a.N.PoutDsrpinQuantity R9376 Product Description Cat. No. a.N.PoutDsrpinQuantity R7647 Product Description Cat. No. Quantity O4387 Product Description Cat. No. Quantity M1302 Product Description Cat. No. synapsin Igene. sequences regulate celltype-specificexpression oftherat (1978). Molecular Biology Manual, 3rdEd., virus reverse transcriptase carboxy-terminal structure ofclonedMoloneymurineleukemia –20 °C –20 °C –20 °C AMV ReverseTranscriptase Random Nonamers Oligo(dT) M-MLV ReverseTranscriptase Biochim. Biophys.Acta, Proc. Natl.Acad.Sci.USA, 50 µM 70 µM Mol. BrainRes. p. 8.48(2001). Meth. Mol.Biol. Biochemistry, 23 Nature, J. Virol., Proc. Natl.Acad.Sci.USA, nhrd0.1ml , Anchored DNA Molec. Cell.Biol., 270, 49, 5, Molecular Cloning:ALaboratory 11, 22, 271-279 (1986). 686 (1984). Totowa, N.J. 314 (1977). 345-353 (1990). 2365 (1985). 473, 1 (1977). 70, Escherichia coli 2, 466 (1973). 161 (1982). 16, 200,000 units 75, 40,000 units 1,000 units 73 (1993). 500 units 1309 0.1 ml of the