RNeasy-Plus-Mini-Kit

Quick-StartProtocolSample&AssayTechnologiesRNeasy®PlusMiniKitTheRNeasyPlusMiniKit(cat.nos.74134and74136)canbestoredatroomtemperature(15–25°C)foratleast9months.Formoreinformation,additionalandmoredetailedprotocols,andsafetyinformation,pleaserefertotheRNeasyPlusMiniHandbook,whichcanbefoundat„IfpurifyingRNAfromcelllinesrichinRNases,ortissue,addeither10μlβ-mercaptoethanol(β-ME),or20μl2Mdithiothreitol(DTT),to1mlBufferRLTPlusbeforeuse.BufferRLTPluscontainingDTTorβ-MEcanbestoredatroomtemperatureforupto1month.„Add4volumesofethanol(96–100%)toBufferRPEforaworkingsolution.„FoamingcanbereducedbyaddingReagentDX(cat.no.19088)atafinalconcentrationof0.5%(v/v)beforedisruptionandhomogenization.**Thisoptionnotincludedinhandbook;handbooktobeupdated.1.Cells:Harvestamaximumof1x107cells,eitherasacellpellet,orlyseddirectlyinthevessel.AddtheappropriatevolumeofBufferRLTPlus(seeTable1).Vortexfor30s,orhomogenize.Tissues:Disruptthetissue(≤30mg)andhomogenizethelysateintheappropriatevolumeofBufferRLTPlus(seeTable1).Centrifugethelysatefor3minatmaximumspeed.Carefullyremovethesupernatantbypipettinganduseitinstep2.2.TransferthehomogenizedlysatetoagDNAEliminatorspincolumnplacedina2mlcollectiontube(supplied).3.Centrifugefor30sat≥8000xg(≥10,000rpm).Discardthecolumn,andsavetheflow-through.Add1volume(usually350μlor600μl)of70%January2011Sample&AssayTechnologiesForup-to-datelicensinginformationandproduct-specificdisclaimers,seetherespectiveQIAGENkithandbookorusermanual.“RNAlater®”isatrademarkofAMBION,Inc.,Austin,TexasandiscoveredbyvariousU.S.andforeignpatents.Trademarks:QIAGEN®,RNeasy®,TissueRuptor®(QIAGENGroup);106755101/2011©2011QIAGEN,allrightsreserved.ethanoltotheflow-through,andmixwellbypipetting.Donotcentrifuge.Proceedimmediatelytostep4.4.Transferupto700μlofthesample,includinganyprecipitate,toanRNeasyspincolumnplacedina2mlcollectiontube(supplied).Closethelid,andcentrifugefor15sat≥8000xg.Discardtheflow-through.5.Add700μlBufferRW1totheRNeasyMinispincolumn(ina2mlcollectiontube).Closethelid,andcentrifugefor15sat≥8000xg.Discardtheflow-through.6.Add500μlBufferRPEtotheRNeasyspincolumn.Closethelid,andcentrifugefor15sat≥8000xg.Discardtheflow-through.7.Add500μlBufferRPEtotheRNeasyspincolumn.Closethelidgently,andcentrifugefor2minat≥8000xg(≥10,000rpm).Optional:PlacetheRNeasyspincolumninanew2mlcollectiontube(supplied).Centrifugeatfullspeedfor1mintofurtherdrythemembrane.8.PlacetheRNeasyspincolumninanew1.5mlcollectiontube(supplied).Add30–50μlRNase-freewaterdirectlytothespincolumnmembrane.Closethelid,andcentrifugefor1minat≥8000xgtoelutetheRNA.Optional:RepeatelutionwithanothervolumeofwaterorwithRNAeluate.Table1.VolumesofBufferRLTPlusforsampledisruptionandhomogenizationSampleAmountDishBufferRLTPlus*Disruptionandhomogenization5x1066cm350μlPelletedcells≤1x1076–10cm600μlAddBufferRLTPlus,vortex(≤1x105cells);oruseQIAshredder,TissueRuptor®,orneedleandsyringe20mg–350μlAnimaltissues20–30mg–600μlTissueLyserLT;TissueLyserII;TissueRuptor,ormortarandpestlefollowedbyQIAshredderorneedleandsyringe*Use600μlBufferRLTPlusfortissuesstabilizedinRNAlater®Reagent,orfordifficult-to-lysetissues.