线虫总rna提取及rt-pcr

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1、C.elegansRNAIsolationandRT-PCRReagentsNeeded:M9(commonstock)Trizol(storedat4ºC)chloroform2-propanol70%EtOHRNase-freeH2OiScriptcDNASynthesisKitAmbionDNA-freeKitBioRadiQSYBRGreenSupermixProcedure:*Wearglovesatalltimes.*Onlyusefiltertips.*Keepeverythingoni

2、ceasmuchaspossibletoavoidRNAdegradation.I.RNAIsolation1.Pick10wormsinto20μLofM9inanRNase-freeEppendorftube.2.Pelletwormsbyspinningbrieflyat14,000rpm.3.Inthehood,add250μLofTrizol.4.Vortexbyhandforabout30secondsandthenvortexat4ºCuntilthewormsdissolve(abou

3、t20minutes)Optional(forlargesamples):a.Letsitatroomtemperaturefor10minutes.*Atthispointthewormscanbefrozen.Removeexcesssupernatantandflashfreezeinliquidnitrogenordryice/EtOH.Storesamplesat-70ºC.b.Centrifugeat14,000rpmfor10minutesat4ºC.c.Transferthesuper

4、natantintoanRNase-freeEppendorftube.5.Inthehood,add50μLofchloroform.6.Vortexfor30seconds.7.Letsitatroomtemperaturefor3minutes.8.Centrifugeat12,000rpmfor15minutesat4ºC.9.Transferthecleartoplayer(~125μL)intoanewRNase-freeEppendorftube.10.Repeatsteps5-9.11

5、.Add125μLof2-propanolandinverttomix.12.Letsitatroomtemperatureforseveralminutes.13.Spindownat12,000rpmfor10minutesat4ºC.14.Carefullydecantthesupernatant.LeaveafewμLsatthebottomsoasnottodisturbthepellet.15.Washpelletwith250-500μLof70%EtOH(useRNase-freeH2

6、O).16.Spindownat14,000rpmfor5minutesat4ºC.17.Removeasmuchsupernatantaspossibleandairdrypelletorusetheairvacuum.18.Dissolvethepelletin10μLofRNase-freeH2O.19.Optional:Heatfor10minutesat60ºCtohelpdissolvetheRNA.Optional:RNA/DNAContaminationQuantification*Y

7、ouwillnotgetanaccuratereadingwithsmallsamples.OD260/OD280:1.8forpureDNA2.0forpureRNASampleshouldbesomewhereinbetween.Makea1:1,000dilution[RNA]=(OD260)x(40μg/mL)x1000[TotalRNA]=[RNA]x19II.DNA-FreeProtocol(AmbionKit)1.Fora20μLreaction,addtoanRNase-freeEpp

8、endorftube:10μLRNA2μL10xDNaseIbuffer7μLRNase-freeH2O1μLDNaseI2.Incubateat37ºC.for20-30minutes.3.Add2.5μLofDNaseInactivationReagent.4.Incubatefor2minutesatroomtemperaturewithoccasionalmixing.5.Spindownat14,000rpmfor1.5minutesat4ºC