Introduction:C.elegansRNAprepsthathavebeenwidelyusedpreviouslyinvolvedfairlyslowlysisoftheworms,potentiallyleADIngtodegradationoftheRNA.Inaddition,theyfailedtoseparateRNAfromgenomicDNA,leadingtodifficultiesindeterminingtheyieldandpurityoftheRNA.Also,thecontaminatingAT-richgenomicDNAmayinterferewithpolyAselectionoftheRNA.ThisprotocolisbasedonstandardmethodsthathavebeenusedformanyyearstoisolateRNAfrommammaliantissuesandDrosophila;itinvolvesveryrapidlysisofthewormsandsubsequentpurificationoftheRNAtohomogeneity.

PrecautionsagainstRNasecontamination:BecauseRNaseisincredIBLystableandiswidelyusedinlabsduringDNApreps,precautionsmustbetakenwhenpreparingsolutions,plasticware,andglasswareforusewithRNAtoavoidcontaminationwiththisenzyme.Glassware(butnottheplasticcaps!)stirbars,andspatulasforweighingreagentsoutshouldbebakedovernightina180oCoven(Horvitzlab:thisisintheautoclaveroom).Plasticware(Pipettetips,centrifugetubes,10mldisposablepipettes)shouldbefromanunopenedpackagethatislabelledRNasefreeandthenkeptinaspecialdrawerafteropening.Mostsolutions,asdetailedbelow,canbetreatedwithdiethylpyrocarbonate(DEPC)toinactivateRNases,andthenautoclavedtoeliminatetheDEPC.DEPCreactswithaminogroups,andthereforecannotbeused,forexample,onTrisbufferedsolutions.AlwayswearcleangloveswhenhandlingRNasefreematerials.Ifatallpossible,useunopenedcleancontainersofreagentstomakeupallsolutions,labelthebottles"RNase free", and store in a special drawer. When weighing out reagents, clean the balance first and use weigh boats from an unopened container.

Solutions:

Caution:WearglovesandavoidgettingDEPConyourskin.

RNasefreewater(makeseveral100mlbottles)

ToddH20,addDEPCto0.1%ShaketogettheDEPCdropletsintosolutionLeaveat37oovernightAutoclave20minutestodestroytheDEPC

1.0MTrispH7.5MakeusingcleantechniqueandDEPCtreatedwaterautoclave

MakeusingcleantechniqueandDEPCtreatedwaterautoclave

MakeusingcleantechniqueandDEPCtreatedwaterautoclave20%sarcosyl(sodiumlaurelsarcosinate)Makeusingcleantechnique,addDEPCto0.1%ShaketogettheDEPCdropletsintosolutionLeaveat37oovernightAutoclave20minutestodestroytheDEPC

Homogenizationbuffer

4.0Mguanidiniumisothiocyanate0.1MTrispH7.5(usetheclean1.0MTrissolutiondescribedabove)sterilefiltertoremoveparticulatematterjustbeforeuse,addB-mercaptoethanolto1%

Cesiumcushionsolution

5.7MCsCl,0.01MEDTApH8Makebymixing96gCsClwith2ml0.5MEDTApH8and~70mlH20Vol.toexactly100ml,sterilefiltertoremoveparticulatematterpourintoabottle,andcarefullymarkthelevelofthemeniscusAddDEPCto0.1%,shaketodissolveDEPCLetstandovernightat37oAutoclave20minutesAfterautoclaving,somevolumemayhavebeenlostduetoevaporation.AddDEPCtreatedwatertobringthemeniscusuptothemark,andmix.

3MsodiumacetatepH5.2

Makeusingcleantechnique,addDEPCto0.1%ShaketogettheDEPCdropletsintosolutionLeaveat37oovernightAutoclave20minutestodestroytheDEPC

RNasefree100%EtOH

Useafreshbottle,thenlabelRNasefreeandkeepclean

RNasefree70%EtOH

Prepareusingtheclean100%EtOHandDEPCtreatedwater

1.Startwithapelletofpurifiedwormsgrowninliquidculture.Itisbesttohave1-5mlsofwormsUSPension(~50%wormsin0.1MNaCl)ina50mldisposablecentrifugetube.Ifyouhavemorethan5mlsofsuspension,thetubewilloverflowwhenyouhomogenize.It"sprobablybettertousefreshlypreparedworms,sincethesecanbelysedmostquickly,butaflashfrozenpelletthatwasstoredat-80oisfine.

2.Usingamotorizedtissuehomogenizeristhefastestmethodtolysetheworms.BylysingquicklyinadenaturingsolutionthatinactivatesRNases,degradationoftheRNAcanbeminimized.We"vebeenusingtheBrinkmannmodel10/35withaPTA10Stip(thisbrandofmachineispopularlyknownasa"polytron"). Be extremely careful when using this machine; it is very powerful and a mishap could be disastrous. The most important safety precaution is to make sure the tip is fastened on the the motor very tightly. During operation the assembly (a big silver nut) that fastens the tip on can start to become unscrewed due to the vibrations; keep your eye glued on the nut and immediately shut the machine off if you see it begin to unscrew! Note: we"re not sure that the polytron will lyse eggs or dauers; this may require sonication.

3.AfteraddingBMEtothehomogenizationbuffer,add5volumesofhomogenizationbuffertothewormsuspension.Ifusingafrozenpelletjustpourthebufferonthefrozenpellet.Immediatelybeginhomogenizing.Withafrozenpellet,startthemachinealowspeedandpushthetipintothepellettobreakitupandreleaseitfromthebottomofthetube.Assoonasthefrozenchunksaregone,turnthemachineuptofullspeed,andhomogenizefor2minutes.(TheoldpolytronfromtheWhiteheadseemstoturnthesolutionblackishbyreleasingsmallmetal?chunks;don"tworry,youcanspintheseout.)

4.Addsarcosylto0.5%(usingtheRNasefree20%solution)andmix.

5.Pourthemixtureintoa28mlpolycarbonateOakRidgetube.Spininthe70Tiultracentrifugerotorfor20minutesat30KRPMat20otopelletdebris(andanymetallicstufffromthepolytron).Pouroffthesupernatantintoaclean50mldisposabletube.Itshouldbebrownishyellow.

6.Youwillneedtoprepareonecesiumgradientforeach6mlsofhomogenate.UseRNasefree16X102mmpolyallomartubes,whichfittheSW28.1buckets(whichintheHorvitzlabwespinonanSW28rotor).Don"tuse"ultraclear" tubes; these crack during the spin. Measure out 11.5 mls of the cesium solution (use a disposable plastic pipette) into each tube. Tap the tubes to eliminate any air bubbles that might be attached to the tube walls within the solution.

7.Drawthewormhomogenateintoa5mlhypodermicsyringefittedwitha23-gaugeneedle.Layerthesampleoverthecesiumpadbyslowlydrizzlingitdownthesideofthetube.Fillthetubeupto1-2mmbelowthetop:thisshouldbe~6mlsofhomogenatepertube.Ifyourunoutofhomogenatewithatubeonlypartlyfilled,youcanfillituptothetopusinghomogenizationbuffer+0.5%sarcosyl.Withafelttippenmakeamarkattheinterfacebetweenthecesiumpadandthehomogenate.

8.CarefullytransferthetubestoSW28.1buckets,screwonthecapsfirmly,carefullyhangthebucketsandmounttherotorintheultracentrifuge,alwaysmakingsurenottodisturbthegradients.Spinat20oCfor24hoursat27KRPM.Useprogram#4(ontheBeckmannL8-80centrifuge)forbothaccelerationanddecelerationsoastodisturbthegradientaslittleaspossiblebeforeandafterthespin.It"sOKtodothespinforafewmoreorlesshoursifnecessary.

9.Afterthespin,carefullyremovethetubesfromthebucketsintoarack,takingcarenotthedisturbthegradient.Thebrownishproteinsshouldstillbeabovethefelttippenmark;youmayseeawhitishdoubletbandwithinthisbrownmaterial.Furtherdownintheclearpartofthecesiumgradientthereshouldbeanotherwhiteband;thisistheDNA.TheRNAisacrystalcleargelatinouspelletLOOSELYattachedtothebottomofthetube;itwillremaininvisibleuntilalmostalltheliquidisremoved.Thepelletlooksaboutlikeawispyflattenedchunkoflowmeltagarose,withavolumeof~30ml.

10.Removingtheliquidrequiresgreatcare,bothtoavoidcontaminatingtheRNAwithmaterialfromhigherupinthegradient,andtoavoidlosingthedifficult-to-seeRNApellet.Usinga10mldisposableplasticpipetteattachedtoarubberbulb,carefullysuckoffthesupernatantdowntothefelttippenmark.Dothisbyholdingthepipettetipattheverysurfaceoftheliquid,sothatbothliquidandairaresuckedup;thisensuresthattheleastdense(andmostcontaminated)materialatthetopofthegradientwillberemovedfirst,andwon"tjustbelowereddowninthetubeasthesolutionbelowissuckedout.Changetoafreshpipette,andsuckoffmoreliquid,inthesamemanner,downtojustbelowthewhitishDNAband.Changepipettes,andsuckmostoftherestoftheliquid,leaving~2mls,sothatliquidjustfillsthecurvedbottompartofthetube.TheremainingliquidwillhavetoberemovedextremelycarefullytoavoidlosingtheRNApellet.

11.Holdanewrazorbladeinabunsenburner(usingforcepsorahemostat)untilitisredhot,andusethebladetocut(reallymelt)throughthetubejustabovetheleveloftheremainingliquid.Itisconvenienttoleaveasmallattachmentbetweentheupperpartofthetubeandthebottomsothatthebottomofthetubecanbecarriedaroundusingtheupperpartasahandle.Thepointofremovingthetopofthetubeistoseparatethecontaminatedwallsofthetubefromthecleanbottomportion,andtohelpyoutoseetheRNApelletinthebottom.

12.UsinganRNasefree200mlpipettetip,VERYCAREFULLYremovetheremainingliquid.ThecleargelatinousRNAmaybefloatingorinseveralpiecesatthispoint;sometimesithelpstotiltthetubebottomaroundtotrytoseparatetheliquidfromthepelletsothattheliquidcanbesafelysuckedoff.Sometimesitistrulyimpossibletoremovethelast~30mlofliquidwithoutsuckingupchunksofRNA.Inthiscase,justleavethelastbitofliquidandaddabout2volumesofroomtemperature100%ethanol(RNasefree).ThiswillcausetheremainingCsCltoformawhiteprecipitatewhich,alongwiththeRNA,willsticktothebottomofthetubebetter.Thenjustgoaheadwiththenextstep(fillingthetubewith70%EtOH).Eventuallyallthiscesiumwillbeeliminatedinthenextethanolprecipitation.

13.Fillthebottomofthetubewithroomtemperature70%EtOH(thissoaksouttheremainingCsCl).TheRNAwillturnwhiteoverthenextcoupleofminutes,andwillattachtothetubemuchmorefirmly.

14.Pourorsuckofftheliquid,anddrythepelletbriefly.WhenthedryingRNAstartstoturnclear,add175mlofTE+0.1%SDStoeachtube,andsuspendtheRNAbypipettingupanddown.Thisshouldtakeabout1minute.TransfertheRNAtoafreshRNasefreescrewcapEppendorftube.Washoutthebottomoftheultracentrifugetubewithanother25mlofTE+0.1%SDS,andcombinewiththerestoftheRNA.IftheRNAisnotcompletelyinsolution(sometimesafewchunksremain)freezeitinliquidnitrogenandthawina50owaterbathonceortwice.

15.Tothe200mlofRNAsolution,add150mlTE(withoutSDS),30ml3MsodiumacetatepH5.2,and900mlEtOH.Shouldseeafluffyprecipitate.Chillat-20ofor30minutes,andmicrofugeinthecoldroomfor10minutes.Discardthesupernatant,washthepelletwith70%ethanol,dry,anddissolveinasmallvolumeofwater,(perhaps~80ml,dependingonhowbigthepelletlooks).

16.MeasuretheconcentrationandpurityoftheRNAbymakinganappropriatedilution(~1000fold)inwaterandtakingtheOD260andOD280.RNApreparedbythismethodshouldbecomletelypure,withanOD260/OD280of2.0.ForcalculatingtheRNAconcentration,asolutionofOD260=1.0is40mg/ml.

16.StoretheRNAat-80o.Dilutingallsamplestoastandardizedconcentration(say5mg/ml)andaliquotingitisagoodidea.

17.Yield:0.8to1.1mgofpuretotalRNApermlofpackedworms.TheseprepswillbecontaminatedtosomeextentwithRNAfromE.colithatwereinthestartingmaterial.TheproportionofE.coliRNAintheprepcanbeassessedbyrunningtheRNAoutonagel,blotting,andstainingtheRNAontheblotwithmethyleneblue;theC.elegansrRNAs,whichrunat3.5and1.7kb,canberesolvedfromtheE.colirRNAs,whichrunat3.0and1.5kb(seeourNorthernblotprotocolfordetailsonthesemethods).Wefoundthatifthewormswerepreparedusingourliquidcultureprotocol,theamountofE.coliRNAinthefinalprepvariedfromundetectabletoabout40%oftheRNA.

================  蚂蚁淘在线  ================

免责声明：本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容

版权声明：未经蚂蚁淘在线授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘在线”。违反上述声明者，本网将追究其相关法律责任。