Description:

 

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.
 
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

Figure2:NEB’sLunaUniversalProbeOne-StepRT-qPCRKitoffersrobustperformanceinmultiplexapplications

Figure3:Extensiveperformanceevaluationofcommerciallyavailableprobe-basedRT-qPCRreagentsdemonstratestherobustnessandspecificityofLuna

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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