RNAGeneralRemarksonHandlingRNARibonucleases(RNases)areverystableandactiveenzymesthatgenerallydonotrequirecofactorstofunction.SinceRNasesaredifficulttoinactivateandevenminuteamountsaresufficienttodestroyRNA,allplasticwareorglasswareshouldbetreatedtoeliminatepossibleRNasecontamination.GreatcareshouldbetakentoavoidinadvertentlyintroducingRNasesintotheRNAsampleduringoraftertheisolationprocedure.InordertocreateandmaintainanRNase-freeenvironment,thefollowingprecautionsshouldbefollowedwhileworkingwithRNA.GeneralhandlingPropermicrobiological,aseptictechniqueshouldalwaysbeusedwhenworkingwithRNA.HandsanddustparticlesmaycarrybacteriaandmoldsandarethemostcommonsourcesofRNasecontamination.AlwayswearlatexorvinylgloveswhilehandlingreagentsandRNAsamplestopreventRNasecontaminationfromthesurfaceoftheskinorfromdustylaboratoryequipment.Changeglovesfrequentlyandkeeptubesclosedwheneverpossible.KeepisolatedRNAonicewhenaliquotsarepipettedfordownstreamapplications.DisposableplasticwareTheuseofsterile,disposablepolypropylenetubesisrecommendedwhenworkingwithRNA.ThesetubesaregenerallyRNase-freeanddonotrequirepretreatmenttoinactivateRNases.Non-disposableplasticwareNon-disposableplasticwareshouldbetreatedbeforeusetoensurethatitisRNase-free.Plasticwareshouldbethoroughlyrinsedwith0.1MNaOH,1mMEDTAfollowedbyRNase-freewater(see“Solutions”,page48).Alternatively,chloroform-resistantplasticwarecanberinsedwithchloroformtoinactivateRNases.GlasswareGlasswareshouldbetreatedbeforeusetoensurethatitisRNase-free.GlasswareusedforRNAworkshouldbecleanedwithadetergent,thoroughlyrinsed,andovenbakedat240°Cforatleast4hours(overnight,ifmoreconvenient)beforeuse.AutoclavingalonewillnotfullyinactivatemanyRNases.Alternatively,glasswarecanbetreatedwithDEPC*(diethylpyrocarbonate).Fillglasswarewith0.1%DEPC(0.1%inwater),incubateovernight(12hours)at37°C,andthenautoclaveorheatto100°Cfor15minutestoeliminateresid-ualDEPC.*DEPCisasuspectedcarcinogenandshouldbehandledwithgreatcare.Wearglovesandu