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NEB/Luna® Universal Probe One-Step RT-qPCR Kit/E3006S/500 reactions
  • NEB/Luna® Universal Probe One-Step RT-qPCR Kit/E3006S/500 reactions

NEB/Luna® Universal Probe One-Step RT-qPCR Kit/E3006S/500 reactions

价格: ¥5700.00 市场价: 9500.00
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货号: E3006S-500reactions
品牌: NEB
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    • Description:

      Sample
       

      Rapid,sensitiveandpreciseprobe-basedqPCRdetectionandquantitationofRNAtargets.

      TheNEBLunaUniversalProbeOne-StepRT-qPCRKitisoptimizedforreal-timequantitationoftargetRNAsequencesusinghydrolysisprobes.One-StepRT-qPCRprovidesaconvenientandpowerfulmethodforRNAdetectionandquantitation.Inasingletube,RNAisfirstconvertedtoCDNAbyareversetranscriptase,andthenaDNA-dependentDNApolymeraseamplifiesthecDNA,enablingquantitationviaqPCR.Probe-basedqPCR/RT-qPCRmonitorsanincreaseinfluorescenceupon5´→3´exonucleasecleavageofaquenched,target-specificprobetomeasureDNAamplificationateachcycleofaPCR.Atapointwherethefluorescencesignalisconfidentlydetectedoverthebackgroundfluorescence,aquantificationcycleorCqvaluecanbedetermined.Cqvaluescanbeusedtoevaluaterelativetargetabundancebetweentwoormoresamples,ortocalculateabsolutetargetquantitiesinreferencetoanappropriatestandardcurvederivedfromaseriesofknowndilutions.

      IntheLunaUniversalOne-StepProbeRT-qPCRKit,HotStartTaqDNAPolymeraseiscombinedwithanovelWarmStart-activatedreversetranscriptase,allowingdualcontrolofenzymeactivityviareversIBLe,aptamer-basedinhibition.Thistemperature-dependentactivationhelpstopreventundesirablenon-specificprimingandextensionpriortoThermocycling,providingaddedsecurityforsettingupreactionsatroomtemperature.TheengineeredWarmStartLunaReverseTranscriptasealsopossesseshigherthermostABIlitythanmanyotherRTs,allowinganoptimalreactiontemperatureof55°C.Fordifficulttargets/templates,higherRTsteptemperaturesofupto60°CcanbeusedwithoutcompromisingLunaperformance.
       
      NotethattoensurefullactivationoftheWarmStartLunaRT,incubationattemperatureslowerthan50°Cisnotrecommended.

      TheLunaUniversalProbeOne-StepReactionMixissuppliedat2XconcentrationandcontainsHot-StartTaqDNAPolymerase,dNTPs,andallrequiredbuffercomponents.Itisformulatedwithauniquepassivereferencedyethatiscompatibleacrossavarietyofinstrumentplatforms,includingthosethatrequireahighorlowROXreferencesignal.TheReactionMixalsofeaturesdUTPforcarryoverpreventionandanon-fluorescentvisibledyeformonitoringreactionsetup.ThisvisibledyedoesnotoverlapspectrallywithfluorophorescommonlyusedinqPCRanddoesnotinterferewithreal-timedetection.

      TheLunaWarmStartRTEnzymeMixissuppliedat20XconcentrationandcontainsLunaWarmStartReverseTranscriptaseaswellasMurineRNaseInhibitortoaidinpreventingRNAdegradation(seealsotemplatepreparationinproductmanual).ItiscompatiblewithvariousRNAsampletypes(totalRNA,poly(A)-RNA,etc.)andsources.

      .

      Figure1:NEB’sLunaUniversalProbeOne-StepRT-qPCRKitoffersexceptionalsensitivity,reproducibilityandRT-qPCRperformance
      RT-qPCRtargetinghumanGAPDHwasperformedusingtheLunaUniversalProbeOne-StepRT-qPCRKitoveran8-lograngeofinputtemplateconcentrations(1μg–0.1pgJurkattotalRNA)with8replicatesateachconcentration.Reactionsetupandcyclingconditionsfollowedrecommendedprotocols,includinga10-minuteRTstepat55°CforthethermostableLunaWarmStartReverseTranscriptase.


      Figure2:NEB’sLunaUniversalProbeOne-StepRT-qPCRKitoffersrobustperformanceinmultiplexapplications
      Luna
      MultiplexRT-qPCRtargetinghumanGAPDH,ribosomalproteinL32gandPI3-Kinase-RelatedKinaseSMG1wasperformedusingtheLunaUniversalProbeOne-StepRT-qPCRKitovera7-lograngeofinputtemplateconcentrations(1μg–1pgJurkattotalRNA)with4replicatesateachconcentration.Amplificationplotsareshownbothoverlayed(left)andforeachmultiplextarget(right).Toaccountforcopynumberdifferences,0.4µMprimerwasusedforlower-copytarget(SMG1)and0.2µMprimerforhigher-copytargets(L32gandGAPDH).Lunamaintainssuperiorefficiency,reproducibility,sensitivityandperformanceinmultiplexRT-qPCR.



      Figure3:Extensiveperformanceevaluationofcommerciallyavailableprobe-basedRT-qPCRreagentsdemonstratestherobustnessandspecificityofLuna
      Luna
      Commercially-availableRT-qPCRreagentsweretestedon7RT-qPCRtargetsvaryinginabundance,length,and%GC.Datawascollectedby2usersandaccordingtomanufacturer’srecommendations.Resultswereevaluatedforefficiency,lowinputdetectionandlackofnon-templateamplification(whereΔCq=averageCqofnon-templatecontrol–averageCqoflowestinput).Inaddition,consistency,reproducibilityandoverallcurvequalitywereassessed(QualityScore).Bargraphindicates%oftargetsthatmetacceptableperformancecriteria(indicatedbygreenboxondotplotandQualityScore>3).ResultsforNEBandothersuppliersareshown:Quanta,qScript™XLT1-StepRT-qPCRToughMix®;ABI,TaqMan®RNA-to-Ct1-StepKit;Qiagen,QuantiFast®ProbeRT-PCRKit;Bio-Rad,iTaq™UniversalProbesOne-StepKit;Promega®,GoTaq®Probe1-StepRT-qPCRSystem.NEB’sLunaUniversalProbeOne-StepRT-qPCRKitoutperformedallotherreagentstested.

      LearnmoreaboutourcomprehensiveqPCR/RT-qPCRtestingand“dotsinboxes”datavisualization.

      KitComponents

      Thefollowingreagentsaresuppliedwiththisproduct:

      Storeat(°C)Concentration
      Luna®UniversalProbeOne-StepReactionMix-202X
      Luna®WarmStart®RTEnzymeMix-2020X
      Nuclease-freeWater-20

      Notes:

      PrimerDesignTheuseofqPCRprimerdesignsoftware(e.g.,Primer3)maximizesthelikelihoodofamplificationsuccesswhileminimizingnonspecificamplificationandprimerdimers.TargetswithbalancedGC/ATcontent(40–60%)tendtoamplifymostefficiently.Wherepossible,entersufficientsequencearoundtheareaofinteresttopermitrobustprimerdesignandusesearchcriteriathatpermitcross-referenceagainstrelevantsequencedatabases(toavoidpotentialoff-targetamplification).ItisadvisabletodesignprimersacrossknownRNAsplicingsitesinordertopreventamplificationfromgenomicDNA.PrimerandProbeConcentrationsFormosttargets,afinalconcentrationof400nM(eachprimer)willprovideoptimumperformance.Ifneeded,primerconcentrationscanbeoptimizedbetween100–900nM.Probeshouldbeincludedat200nMforbestresults.Probeconcentrationcanbeoptimizedintherangeof100–500nM.MultiplexingWhendeterminingwhichfluorophorestoincludeinamultiplexreaction,besuretochoosecompatiblereporterdyesandquenchers(e.g.,thosethatcanbeaccommodatedbythechosenreal-timeinstrumentwithminimaloverlapinfluorescencespectra).ForROX-dependentinstruments,avoidROX-labeledprobes.Include400nMofforwardandreverseprimersand200nMprobeforeachtargettobedetectedinthereaction.Fortargetsthatdiffersignificantlyinabundance,useofalowerprimerconcentration(e.g.200nM)forthemoreabundanttarget(s)isrecommended.Adjustconcentrationsifnecessarybasedonperformance(primer100–900nM,probe100–500nM).WhenloADIngaqPCRprotocolontothereal-timeinstrument,besuretoselecttheappropriateopticalchannels,assomeinstrumentshaveasinglechannelrecordingmodethatwouldpreventmultiplexdatacollectionandanalysis.Thefunctionalityoftheprimerandprobesetsshouldbetestedindividuallybeforeattemptingamultiplexreaction.AmpliconLengthToensuresuccessfulandconsistentqPCRresults,itisimportanttomaximizePCRefficiency.AnimportantaspectofthisisthedesignofshortPCRamplicons(typically70–200bp).Someoptimizationmayberequiredfortargetsthatexceedthatrange.TemplatePreparationandConcentrationLunaRT-qPCRiscompatiblewithRNAsamplespreparedthroughtypicalnucleicacidpurificationmethods.PreparedRNAshouldbestoredinanEDTA-containingbuffer(e.g.,1XTE)forlong-termstability,anddilutionsshouldbefreshlypreparedforaqPCRexperimentineitherTEorwater.NotethatthequalityofRNAtemplatescangreatlyaffectRT-qPCRefficiency.RNAshouldbehandledwithappropriateprecautionstopreventRNaseorDNasecontamination.Useofnuclease-freewater(provided)isstronglyrecommended.Whereuseful,RNAmaybetreatedwithDNaseItoremovecontaminatinggenomicDNA.Generally,ausefulconcentrationofstandardandunknownmaterialwillbeintherangeof108copiesto10copies.Notethatfordilutionsinthesingle-copyrange,somesampleswillcontainmultiplecopiesandsomewillhavenone,asdefinedbythePoissondistribution.FortotalRNA,LunaOne-StepKitscanprovidelinearquantitationoveran8-orderinputrangeof1μg–0.1 pg.Formosttargets,astandardinputrangeof100ng–10pgtotalRNAisrecommended.ForpurifiedmRNA,inputof≤100ngisrecommended.Forinvitro-transcribedRNA,inputof≤109copiesisrecommended.ROXReferenceDyeSomereal-timeinstrumentsrecommendtheuseofapassivereferencedye(typicallyROX)toovercomewell-to-wellvariationsthatcouldbecausedbybubbles,smalldifferencesinvolume,andautofluorescencefromdustorparticulatesinthereaction.LunamixesareformulatedwithauniversalreferencedyethatiscompatiblewithavarietyofqPCRinstrumenttypes,includingthosethatusenopassivereferencenormalizationandthosethatusealoworhighconcentrationofpassivereferencedye(ROX).Therefore,noadditionalcomponentsarerequiredtoensurecompatibilitywiththeseinstruments.CarryoverContaminationPreventionRT-qPCRisanextremelysensitivemethod,andcontaminationinnewRT-qPCRassayswithproductsfrompreviousamplificationreactionscancauseavarietyofissues,suchasfalsepositiveresultsandadecreaseinsensitivity.Thebestwaytopreventthis“carryover”contaminationistopracticegoodlaboratoryproceduresandavoidopeningthereactionvesselpostamplification.However,toaccommodatesituationswhereadditionalanti-contaminationmeasuresaredesired,LunaqPCRmixescontainsamixtureofdUTP/dTTPthatresultsintheincorporationofdUintotheDNAproductduringamplification.PretreatmentofqPCR/RT-qPCRexperimentswithuracilDNAglycosylase(UDG)willeliminatepreviously-amplifieduracil-containingproductsbyexcisingtheuracilbasetoproduceanon-amplifiableDNAproduct.TheuseofathermolabileUDGisimportant,ascompleteinactivationoftheUDGisrequiredtopreventdestructionofnewlysynthesizedqPCRproducts.Toenablecarryoverprevention,0.025units/μlAntarcticThermolabileUDG(NEB#M0372)shouldbeaddedtothereactionmix.Tomaximizeeliminationofcontaminatingproducts,setuptheqPCRexperimentsatroomtemperatureorincludea10minuteincubationstepat25°Cbeforetheinitialdenaturationstep.ReactionSetupandCyclingConditionsDuetodualhot-startfeatureofLunaOne-StepKits,itisnotnecessarytosetupreactionsoniceorpreheatthethermocyclerpriortouse.For96-wellplates,afinalreactionvolumeof20μlisrecommended.For384-wellplates,afinalreactionvolumeof10μlisrecommended.Whenprogramminginstrumentcyclingconditions,ensureaplatereadisincludedattheendoftheextensionstep,andadenaturation(melt)curveaftercyclingiscompletetoanalyzeproductspecificity.Amplificationfor40cyclesissufficientformostapplications,butforverylowinputsamples45cyclesmaybeused.
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