PreparationofPolyA+RNAandNorthernAnalysis

Procedure(Pharmacia#27-9255-01)

1)RemoveorgansfromsixC57Blmice(docervicaldislocationimmediatelybeforeorganremoval)andfreezedirectlyintoliquidnitrogen.Storeat-70°C.Forlacrimalgland,inwhichmRNAyieldsdirectlyfromtheorganhaveoftenbeenlow,abetterapproachistodoanisolationoflacrimalacinarcellsfromsixC57Blmice(seemethodinLabProtocols;leaveoutPercollstep)first,thenextractmRNA.Toextractfromculturedcells(ie.HT1080),beginwithoneT75flask(approx.2x106cells).

2)HavePharmaciakitreagentswarmedtoRTfor30min.CheckifExtractionbufferisinsolution;ifnotwarmto37°CthencooltoRT.Havewaterbathat65°Cand0.5ml/purificationofelutionbufferwarmingat65°C.ResUSPendOligo(dT)andpipet1mlaliquotsintoindividualmicrocentrifugationtubes(1ml/purification).Weighout0.1-0.23gmoftissue,andplaceina7mlmortar(alternatively,canuseapolytron).Add0.4mlofextractionbuffer;usepestletohomogenize.Add0.8mlofelutionbuffer,mix,homogenizeandtransfertoaclean1.5mlmicrocentrifugationtube.Centrifugefor1min(RT)fullspeed.CentrifugeOligo(dT)for2sec(justbeforegetstofullspeed,releasebutton;spinninglongermakesresuspensionofOligo(dT)difficult).DiscardsupernatantfromOligo(dT)andaddtoit1mlofsupernatantfromtissuehomogenization.Mixfor3minbyinvertingonrotor.

3)Spin2sec,discardsupernatant.Washfivetimeseachwith1mlofHighSaltBuffer(quickspinsbetweenseveralinversionstomix).Washtwotimeseachwith1mlofLowSaltBuffer.Resuspendin0.3mlofLowSaltBufferandtransfertoaMicrospinColumn(breakoffendbeforeuse)inamicrocentrifugetube.Spinatfullspeedfor5sec.Discardeffluentandadd0.5mlofLowSaltBuffer.Spinandwashtwomoretimeswith0.5mleachofLowSaltBuffer.Toelute,placecolumninafreshmicrocentrifugetubeandadd0.2mlofprewarmedElutionbuffer.Spinatfullspeedfor5sec.Addasecond0.2mlofprewarmedElutionbufferandspinagain.PlaceelutedmRNAonice.

4)TodetermineOD260ofmRNA,haveaquartzcuvettesoakinginconc.HCl/methanol(1:1)for1hrpriortousetoremoveRNase.RinsewellwithDEPCtreatedddH2O.ZerospectrophotometerwithelutionbufferthenaddallofmRNAsampletocuvettetodetermineOD260andOD260/OD280.Forµg/mlRNA,multiplyOD260valuetimes40(1OD260is40µg/ml).Forstorage,precipitatemRNAwith10µlofGlycogensolution,40µlofpotassiumacetatesolutionand1mlof-20°C95%EtOH.Storeat-70°C.

5)ForNorthernanalysis,haveheatingblockat56°Candpourdenaturinggel(mid-sizegel:to0.8gmofagarose,add78.4ddH2Oand5mlof20xEbuffer;heattodissolve;letcooltonottoohottotouch,thenquicklyadd16.6mlof37%formaldehyde,swirltothoroughlymixandpour).SpinprecipitatedmRNAfor15min(4°C,fullspeed).Carefullyremoveanddiscardsupernatant.WashpelletwithRNase-free80%EtOH,dryandmakeupin20µlof"BufferA".AlsohavesizestandardsincludinganRNAladder(mix20µlofladder+28.5µlof"SampleBuffer")and28S/18SrRNAstandard(mix20µlofladder+28.5µlof"SampleBuffer").Heatat56°Cfor15min,thenplaceonice.Add4.9µlof"GlycerolStain"tomRNAand12.5µltostandards.Loadontogelimmersedin1xE/formaldehydebuffer(seebelow)withstandardsintheoutsidelanes.Runovernightat35V(constant).

20XEBuffer

Na2HPO4.7H2O32.72gm

NaH2PO4.H2O10.85gm

ddH2Oto1000ml

1xE/FormaldehydeBuffer

ddH2O773ml

20xEbuffer50ml

37%formald.177ml

SampleBuffer

formamide145.2µl

20xEbuffer12.8µl

37%formald.42µl

BufferA

DEPCddH2O118µl

samplebuffer200µl

GlycerolStain

DEPCddH2O7ml

glycerol3ml

bromophenolbl.25mg

xylenephenol25mg

DEPCWater

Add0.8mlofDEPC(Sigma#D-5758)to800mlddH2O.Warmovernightat37°thenautoclave.

6)Carefullycutoutthestandardslanesandplaceinethidiumbromide(5µg/ml;5µlofstockper100mlofddH2O)for30min.WashovernightinddH2O,thenphotographontransIlluminatorwithflorescentruler.Therestofthegel(cutofftheleftbottomcornertoaidinorientation)istransferredtoacleanglasstrayandimmediatelydenaturedin900mlof50mMNaOH/10mMNaCl(to893.7mlofddH2Oadd4.5mlof10NNaOHand1.8mlof5MNaCl)for20minonarotator.Carefullypouroffandadd900mlof0.1MTris,pH7.6toneutralize(20minonrotator).Pouroffandadd900mlof20xSSCfor45minwithgentlerotation.Tokeepgelimmersedduringabovesteps,canbalanceaninvertedddH2Ofilled50mlconicalonit,makingsurethatthetopsurfaceofthegeliswetbeforeplacingontheconical.

7)Duringtheabove20and45minsteps,preparationsfortheovernighttransfercanbeinitiated.Neededare:(i)aglasstrayorluciteboxfilledtoadepthof2cmwith10xSSC,(ii)alucitesupportforthegel(caninvertthegelmoldinthereservoir),(iii)alongpieceof3MMpaperwhichstretchesoverthelucitegelsupportoneithersideintothe10xSSC;wetwith10xSSC,(iv)nitrocellulose(AmershamHybondECL)cuttothesamesizeasthegel(wearglovesinthisandallsteps),(v)twopiecesof3MMpapercuttothesamesizeasthegel;placeoneonthelong3MMpieceandwetwith10xSSC,(vi)a6-8inchstackofpapertowelscuttothesamesizeasthegel,(vii)aglasstraycontainingddH2O,(viii)aglasstraycontaining10xSSC,(ix)two10Óx12ÓpiecesofX-rayfilm,and(x)flatforceps.

8)Tosetupthetransfer,sandwichthegelbetweentheX-rayfilmpiecesandcarefullyinvert(dooverthe20xSSC).Placebacksideuponthewettedgel-size3MMpaper(oninvertedmold).Removeanyairbubblesbygentlyrollingacapless50mlconicaloverthegel.Addsome10XSSCtokeepfromdryingout.CompletelysurroundthegelwithParafilmorplasticwrap.Next,wetthenitrocellulosepaperbyfloatingon,thenimmersingin,theddH2O.Immersethewetfilterin10xSSCfor5min.Holdingthefilterbytwosides,carefullysetdownexactlyonthegel(canÕtreposition).Carefullyrollthecaplessconicaloverthefiltertoensureremovalofanyairbubbles.Addtheremaininggel-sizepieceof3MMpaper(wetwith10xSSC)androllwithconical.Carefullyaddthecutpapertowelstoaheightof6Ó.Balanceaglasstrayontopofthetowelstoweighdown;stABIlizethetraywithtaperunningdownonallsidestothe10xSSCreservoir.Coverwithplastictoreducelosstoevaporation.Iftransferisinitiatedinthemorning,carefullychangethepapertowels(canreusethetopdryones)beforeleavingattheendoftheday.Letthetransfergoovernight.

9)Have80°Cvacuumovenheatingup.Removethepapertowelsand,withthegel/filterstillinplace,invert.UseanindelIBLefinetipMarkertomarkonthefiltertheedgesofthegel(includingthecutcorner)andtheloADIngwells.Recordexperimentalnumberatthetop.Peeloffthegelandstainfor45minin0.5µg/mlethidiumbromide(thenplaceinddH2Oandexamine/photographontransilluminator).Placefilterin6xSSCfor5mintowash,thenplaceon3MMpaperanddryforatleast30min.Placethedriedfilterbetweentwopiecesof3MMpaperandbakeundervacuumfor1-2hr.

10)Tocarryouthybridizationtofilter,prehybridizefilterfor4hrat42°C(inprehyb/hyb.plasticboxes;gentlyrotation)inaprewarmedsolutionof30%formamide,5xSSPE,0.1%SDS,5xDenhardtÕs,0.2mg/mlsonicateddenaturedsalmonspermDNA.

PrehybridizationSolÕnHybridizationSolÕnddH2O15.3mlddH2O15.3mlFormamide15mlFormamide15ml20xSSPE12.5ml20xSSPE12.5ml20%SDS0.25ml20%SDS0.25ml50xDenhardtÕs5ml50xDenhardtÕs5mlSalmonSp.DNA2mlSalmonSp.DNA2mlPolyA5µlPolyA5µl32CDNAprobe50-100µl

(note:SalmonSpermDNAstockis5mg/ml;PolyAstockis10mg/ml;makeupprehybandhybsol"nsin50mlconicals)

11)Duringprehybridization,labelcDNAprobe(purifiedinsert)byrandomprimingmethod(Pharmacia#27-9251-01).Reconstitutereactionmixbyadding20µlofddH2Oandplaceonicefor5-60min.Make1.8µlofinsertupin25µlofTE,pH8anddenaturefor3minat95°C.Coolonicefor2min,thenadddiluted/denaturedinserttoreactionmix.Behindlucite,add5µlof32PdCTP(ICN#33004X).Carefullymixbypipettingupanddown.Incubateat37°Cfor15-30min.PurifyprobeusingaNickSpinColumn(Pharmacia#17-0862-02)orequivalent(Qiagen#28304)andcheckcpmÕsinScintillationcounter.Denatureprobe(3minat95°C,onicefor2min)priortoadditiontohybridizationsolution.Hybridizeovernightat42°Cwithgentlyrotation.

12)Removefilterinto400mlofroomtemperature2xSSC,0.1%SDS(40mlof20xSSCand4mlof20%SDSmadeupto800mlinddH2O).Washfor5minonrotator.Dotwoadditionalwashes.Transferfilterto400mlofprewarmed(42-65°C;use42°Cforwheat-mouseand65°Cformouse-mousehybridizations)0.1xSSC,0.1%SDS(4mlof20xSSCand4mlof20%SDSmadeupto800mlinddH2O).Washfor20minonrotator.Doatotalofthree20minwashes.

13)Allowfiltertodryonfreshbenchpaper,thenplaceonWhatmann3MMpaperinalargex-raycassetteholder.Coverwithplasticwrap.Underredlightinthedarkroom,placex-rayfilmon,followedbyanenhancingscreen.Closeupandexposeovernightat-70°C.