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.