Omega公司RNA提取试剂盒说明书

RNA-SolvReagent®RNAIsolationSolventWARNING:Thisreagentistoxicifswallowedandincontactwithskin.Causesburns.Aftercontactwithskin,washimmediatelywithcopiousamountsofmilddetergentandwater.Ifyoufeelsick,seekmedicaladviceatonceandQuoteUN2821.ProductNo:R6830-00(5ml)R6830-01(100ml)R6830-02(200ml)StorageConditions:RNA-Solvisstableforatleast24months®whenstoredat2°C-8°Candyieldsreproducibleresults.IntroductionRNA-Solv®ReagentisareagentsystemfortheisolationoftotalRNAfromcellsandtissues.Thereagent,asingle-phasesolutionconsistingofphenolandguanidineisothiocyanate,ismodificationofthesingle-stepRNAisolationmethoddevelopedbyChomczynskiandSacchi(1).ThesampleishomogenizedandlysedinRNA-Solv®ReagentwhichmaintainstheintegrityoftheRNA,whiledisruptinganddenaturingendogenousRNasesandothercellularcomponents.Extractionofthelysatewithchloroformfurtherdenaturesproteinsandseparatesthemixtureintoanorganicandanaqueousphase.RNAremainsexclusivelyintheaqueousphase,andissubsequentlyrecoveredbyisopropanol.Thismethodissuitableforsmallquantitiesoftissue(100mg)andcells(5X10),andlargequantitiesoftissue(upto1g)andcells6(10),ofhuman,animal,plant,orbacterialorigin.Thesimplicity8oftheRNA-Solv®Reagentmethodallowssimultaneousprocessingofalargenumberofsamples.Theentireprocedurecanbecompletedinonehour.TotalRNApreparedinthismannercanbeusedforNorthernblotanalysis,dotblothybridization,poly(A)+selection,invitrotranslation,RNaseprotectionassay,andmolecularcloning.Foruseinamplificationbythermalcycling,treatmentoftheisolatedRNAwithRNase-freeDNaseIisrecommendedwhenthetwoamplimersliewithinasingleexon.SuppliedByUser•Chloroform(noisoamylalcoholadded)•Isopropylalcohol•80%Ethanol(inDEPC-treatedwater)•RNase-freewater•Tabletopcentrifugecapableof12,000xgatroomtemperatureGeneralNotesRegardingRNaseContaminationWheneverworkingwithRNA:•Alwaysweardisposableglovesandchangeglovesfrequently.•Usesterile,disposableplasticwareandautomaticpipettesreservedforRNAworktopreventcross-contaminationwithRNases.•InthepresenceofRNA-Solv®Reagent,RNAisprotectedfromRNasecontamination.DownstreamsamplehandlingrequiresthatnondisposableglasswareorplasticwarebeRNase-free.•UseonlyDECP-treatedbuffers.AddDEPCtoafinalconcentrationof0.1%,incubateat37Cfor2hours,andoautoclaveat121C.DonotaddDEPCtoTrisbuffers.SuchobuffersmustbepreparedbyusingDECP-water.PrecautionUseonlydisposablepolypropylenetubesforsmallsamplesandglassCorextubesforlargersamples.Alltubesmustbeabletowithstand12,000xg.PolystyrenetubesmaycrackwithchloroformBeforeStartingA.SmallSamples:ToisolateRNAfromverysmallsamples(106cellsor10mgtissue)performhomogenization(orlysis)ofsamplesin0.8mLofRNA-Solv®,andadd1mgRNase-freeglycogenoryeasttRNAascarrier.Thiswillimproveyieldsobtainedwithprecipitation.B.DifficultAnimalSamples:Specimenscontaininglargeamountsofproteins,fat,polysaccharidesorextracellularmaterialsuchasmuscles,fattissue,andsperm,willrequirethefollowingmodification.Afterlysis/homogenizationinRNA-Solv®Reagent,centrifugeat12,000xgfor10minutesatroomtemperaturetoremoveinsolubledebris.Oftenaprecipitateformsatthebottomofthetube,butwithfattytissue,alipidlayerwillalsoformabovetheaqueousphase.ThesupernatantwillcontaintheRNAandmustbecarefullytransferredtoafresh1.5mlmicrofugetubebeforeproceeding.C.Interruptiontheprocedure:FollowinglysisinRNA-Solv®Reagentandbeforeadditionofchloroform,samplescanbestoredat-70Cforupto3months.Inaddition,oncetheRNAisoprecipitatedinisopropanol,thepelletmaybestoredat-20Cor-o70Cforupto1year.oRNA-SolvProtocolforTotalRNAIsolation®CAUTION:WhenworkingwithRNA-Solv®Reagentuseglovesandeyeprotection(safetygoggles)andavoidcontactwithskinorclothing.Workinachemicalfumehoodtoavoidinhalingvapor.Unlessotherwisenoted,allstepsaretobecarriedoutatroomtemperature(20C-25C).oo1.Homogenizationandlysisofsamples:followeithermethodbelowa)TissueSamplesHomogenizetissuesamplesin1mLofRNA-Solv®Reagentper50-100mgoftissueusinganappropriatemechanicalhomogenizer.Alternativelyonecanpulverizetissueinliquidnitrogenwithmortarandpestleandtransferthepowdertoaclean1.5mlmicrocentrifugetube.Ifceramicmortarandpestlearenotavailable,homogenizethesampleinthemicrofugetubeusingadisposablemicrotubepestle(Eppendorf,CatNo.0030120.973;VWR,CatNo.KT749520-0000).Thesamplevolumeshouldnotexceed10%ofthevolumeofRNA-Solv®Reagentused.b)CellsGrowninSuspensionPelletcellsbycentrifugation.LysecellsinRNA-Solv®Reagentbyrepetitivepipetting.Use1mLofthereagentper5-10x10of6animal,plantoryeastcells,orper1x10bacterialcells.Washing8cellsbeforeadditionofRNA-Solv®ReagentshouldbeavoidedasthisincreasesthepossibilityofmRNAdegradationandRNasecontamination.Forplant,fungal,andyeastcellsmechanicalorenzymatichomogenizationmayberequired.Also,forplant,fungal,andyeastcells,werecommendtheuseoftheE.Z.N.A.®Plant(R6627),Fungal(R6640),andYeast(R6670)RNAKitsfromOmegaBio-tek.c)CellsGrowninMonolayerLysecellsdirectlyinaculturedishbyadding1mLofRNA-Solv®Reagenttoa3.5cmdiameterdish,andpassingthecelllysateseveraltimesthroughabluepipettetip.TheamountofRNA-Solv®Reagentaddedisbasedontheareaoftheculturedish(~1mLper10cm).Aninsufficien