microRNA定量PCR检测方法

好将PCR产物进行电泳检测产物是否单一（因产物长度很小，需要3%以上的琼脂糖胶）。3）探针设计（primerexpress）：◆TaqMan探针位置尽可能靠近扩增引物(扩增产物50-150bp)，但不能与引物重叠。◆长度一般为18-40mer（最好是20-30bp）。◆避免连续相同碱基的出现，特别是要避免GGGG或更多G出现。◆在引物的5’端避免使用G--因为5'G会有淬灭作用，而且即使是被切割下来还会存在淬灭作用。可选用比较多的碱基C。◆退火温度TmTm值在65-70℃，通常比引物TM值高5-10℃（至少要5℃），GC含量在40%－70%。实例参考：RT-PCR引物及探针设计：Real-timequantificationofmicroRNAsbystem–loopRT–PCR,CaifuChenetal.NucleicAcidsResearch,2005,Vol.33,No.20e179ReversetranscriptasereactionscontainedRNAsamplesincludingpurifiedtotalRNA,celllysate,orheat-treatedcells,50nMstem–loopRTprimer(P/N:4365386and4365387,AppliedBiosystems),1xRTbuffer(P/N:4319981,AppliedBiosystems),0.25mMeachofdNTPs,3.33U/mlMultiScribereversetranscriptase(P/N:4319983,AppliedBiosystems)and0.25U/mlRNaseinhibitor(P/N:N8080119;AppliedBiosystems).The7.5mlreactionswereincubatedinanAppliedBiosystems9700Thermocyclerina96-or384-wellplatefor30minat16C,30minat42C,5minat85Candthenheldat4C.AllReversetranscriptasereac-tioincludingno-templatecontrolsandRTminuscontrols,wereruninduplicate.Real-timePCRwasperformedusingastandardTaqManPCRkitprotocolonanAppliedBiosystems7900HTSequenceDetectionSystem(P/N:4329002,AppliedBiosystems).The10mlPCRincluded0.67ulRTproduct,1xTaqManUni-versalPCRMasterMix(P/N:4324018,AppliedBiosystems),0.2uMTaqManmprobe,1.5uMforwardprimerand0.7uMreverseprimer.Thereactionswereincubatedina384-wellplateat95Cfor10min,followedby40cyclesof95Cfor15sand60Cfor1min.Allreactionswererunintriplicate.Thethreshcycle(CT)isdefinedasthefractionalcyclenumberatwhichthefluorescencepassesthefixedthreshold.TaqManCTvalueswereconvertedintoabsolutecopynumbersusingastandardcurvefromsyntheticlin-4miRNA.血清血浆中miRNA定量检测方法：AnalysisofcirculatingmicroRNAbiomarkersinplasmaandserumusingquantitativereversetranscription-PCR(qRT-PCR),EvanM.Krohetal.Methods502(2010)298–3012ReversetranscriptionReversetranscriptionreactionsareperformedusingtheTaq-ManmiRNAReverseTranscriptionKitandmiRNA-specificstem-loopprimers(PartNo.4366597,AppliedBioSystems,Inc.)inascaleddown(5lL)RTreaction:Eachreactionshouldbecomprisedof1.387uLH2O,0.5u10xReverse-TranscriptionBuffer,0.063uLRNase-Inhibitor(20U/uL),0.05uL100mMdNTPswithdTTP,0.33uLMultiscribeReverseTranscriptase,1uLRTprimer.Thesecomponentsshouldbepreparedasalargermastermix.Mixbyinversion(donotvortex),andcollectcontentsbybriefcentrifugation.Aliquotmastermixinto0.2mLRNase-freestriptubesora96-wellplateandadd1.67uLinputRNA.Forgenerationofstan-dardcurvesusingchemicallysynthesizedRNAoligonucleotidescorrespondingtoknownmiRNAs,seriallydilutethesyntheticmiRNAsasdescribedinSection2.8andaddtoRTreactionsatavolumeof1.67uLperreaction.MixRTreactionsbyinversionandcentrifugetocollectcontents.UseaTetrad2PeltierThermalCycler(BioRad)tocarryouttheRTreactionsusingthefollowingconditions:16Cfo30min,42Cfor30min,85Cfor5min,holdat4C.RTproductscanbestoredundilutedatCpriortorunningthereal-timePCR.Real-timePCRReal-timePCRreactionsareperformedinduplicate,inscaled-down(5uL)reactionvolumesusing2.5uLTaqMan2xUniversalPCRMasterMixwithNoAmpEraseUNG,0.25uLmiRNA-specificprimer/probemix,and2.25uLdilutedRTproductperreaction.ForeachmiRNA-specificassay,prepareareactionpre-mixbycombiningsufficientTaqMan2xUniversalPCRMasterMixandprimer/probemixforallreactions,plusexcessforlossesassociatedwithpipetting.Mixbyinversionandcentrifugebriefly.Aliquotenoughreactionmixfortwoduplicatereactionspersampleintostriptubes(i.e5.5uLreactionmixplus15%excessforpipettinglosses,peraliquot,willbesufficientforduplicatereactionsforasample,because2.75uLreactinpre-mixisneededperfinalreaction).DilutetheRTproductsbycombining5.0uLRTproductwith28.9uLwater(1:15finaldilutioninPCRreaction).Mixandcentrifugebriefly.Foreachsample,addthedilutedRTproducttothereactionpremixaliquotsfromstep2,addingenoughforduplicatereactions(i.e.,4.5lLdilutedRTproductplus15%excess,because2.25uLdilutedRTproductisneededperPCRreaction).Mixtheduplicatereactions,centrifuge,andaliquot5uLreactionsinduplicateintotheopticalplate.SealwithABIMicroAmp™OpticalAdhesiveFilm.Centrifugeplatetoensurenobubblesinhibitsignaldetection.Real-timePCRiscarriedoutonanAppliedBioSystems7900HTthermocycler(AppliedBiosystems,Inc.)usingthefollowingconditions:95Cfor10min,followedby40cyclesofCfor15sand60Cfor1min,