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NEB/Luna? Universal Probe One-Step RT-qPCR Kit/E3006S/200 reactions

價格: ￥2532.00 市場價: 4220.00

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貨號: E3006S-200reactions

品牌: NEB

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- Description:
  
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  Rapid,sensitiveandpreciseprobe-basedqPCRdetectionandquantitationofRNAtargets.
  TheNEBLunaUniversalProbeOne-StepRT-qPCRKitisoptimizedforreal-timequantitationoftargetRNAsequencesusinghydrolysisprobes.One-StepRT-qPCRprovidesaconvenientandpowerfulmethodforRNAdetectionandquantitation.Inasingletube,RNAisfirstconvertedtoCDNAbyareversetranscriptase,andthenaDNA-dependentDNApolymeraseamplifiesthecDNA,enablingquantitationviaqPCR.Probe-basedqPCR/RT-qPCRmonitorsanincreaseinfluorescenceupon5′→3′exonucleasecleavageofaquenched,target-specificprobetomeasureDNAamplificationateachcycleofaPCR.Atapointwherethefluorescencesignalisconfidentlydetectedoverthebackgroundfluorescence,aquantificationcycleorC_qvaluecanbedetermined.C_qvaluescanbeusedtoevaluaterelativetargetabundancebetweentwoormoresamples,ortocalculateabsolutetargetquantitiesinreferencetoanappropriatestandardcurvederivedfromaseriesofknowndilutions.
  
  IntheLunaUniversalOne-StepProbeRT-qPCRKit,HotStartTaqDNAPolymeraseiscombinedwithanovelWarmStart-activatedreversetranscriptase,allowingdualcontrolofenzymeactivityviareversIBLe,aptamer-basedinhibition.Thistemperature-dependentactivationhelpstopreventundesirablenon-specificprimingandextensionpriortoThermocycling,providingaddedsecurityforsettingupreactionsatroomtemperature.TheengineeredWarmStartLunaReverseTranscriptasealsopossesseshigherthermostABIlitythanmanyotherRTs,allowinganoptimalreactiontemperatureof55°C.Fordifficulttargets/templates,higherRTsteptemperaturesofupto60°CcanbeusedwithoutcompromisingLunaperformance.
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  NotethattoensurefullactivationoftheWarmStartLunaRT,incubationattemperatureslowerthan50°Cisnotrecommended.
  
  TheLunaUniversalProbeOne-StepReactionMixissuppliedat2XconcentrationandcontainsHot-StartTaqDNAPolymerase,dNTPs,andallrequiredbuffercomponents.Itisformulatedwithauniquepassivereferencedyethatiscompatibleacrossavarietyofinstrumentplatforms,includingthosethatrequireahighorlowROXreferencesignal.TheReactionMixalsofeaturesdUTPforcarryoverpreventionandanon-fluorescentvisibledyeformonitoringreactionsetup.ThisvisibledyedoesnotoverlapspectrallywithfluorophorescommonlyusedinqPCRanddoesnotinterferewithreal-timedetection.
  
  TheLunaWarmStartRTEnzymeMixissuppliedat20XconcentrationandcontainsLunaWarmStartReverseTranscriptaseaswellasMurineRNaseInhibitortoaidinpreventingRNAdegradation(seealsotemplatepreparationinproductmanual).ItiscompatiblewithvariousRNAsampletypes(totalRNA,poly(A)-RNA,etc.)andsources.
  
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  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
  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
  Commercially-availableRT-qPCRreagentsweretestedon7RT-qPCRtargetsvaryinginabundance,length,and%GC.Datawascollectedby2usersandaccordingtomanufacturer’srecommendations.Resultswereevaluatedforefficiency,lowinputdetectionandlackofnon-templateamplification(whereΔC_q=averageC_qofnon-templatecontrol–averageC_qoflowestinput).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 -20 2X
  Luna^?WarmStart^?RTEnzymeMix -20 20X
  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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NEB/Luna? Universal Probe One-Step RT-qPCR Kit/E3006S/200 reactions

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KitComponents

Notes:

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	Storeat(°C)	Concentration
Luna^?UniversalProbeOne-StepReactionMix	-20	2X
Luna^?WarmStart^?RTEnzymeMix	-20	20X
Nuclease-freeWater	-20