NorthernBlots

byMichaelKoelleandToryHerman,adaptedfromSambrooketal.,"MolecularCloning"

4/6/94

WefoundthatbothformaldehydeandglyoxalgelsworkverywellforelectrophoresisofRNA;weonlypreferglyoxalgelsbecausethefumesfromtheformaldehydegelsareunpleasant.Mostprotocolssuggestusingnitrocelluloseratherthanchargednylonmembranesforblotting;thelatteraresaidtogivehigherbackground.Wehavebeenusingunchargednylon("Nytran"fromSchleicherandSchuell)withgoodresults.BecauseagoodnylonNorthernblotcanbekeptforyearsandreprobedadozenormoretimeswithgoodresults,itseemsashametousenitrocellulose,whichissophysicallyweakthattheblotisunlikelytolastmorethanacoupleofprobingswithouttearing.

AswithallRNAwork,precautionsagainstRNAsecontaminationshouldbetaken.SolutionsshouldbeDEPCtreated;reagentsandplasticwareshouldbefromfreshpackagessetasideforRNAwork;glassware,spatulas,stirbars,etc.shouldbedecontaminatedbybakingovernightat180deg.C.BecauseelectrophoresisboxesareoftenusedtoanalyzeDNAsamplesthathavebeenRNasetreated,theyareofspecialconcern.AspecialgelboxandcombsshouldbesetasideforRNAworkonly.Ifthereissomequestionthatagelboxorcombhasbeencontaminated,theycanbecleanedbythoroughwashingwithdetergent,rinsing,soakingwith3%hydogenperoxidefor10minutesatroomtemperature,andfinallyrinsinginDEPCtreatedwater.

Solutions:

Caution:WearglovesandavoidgettingDEPConyourskin.

RNasefreewater(makeseveralliterstomakeuptherunningbuffer.Havingsome100mlbottlesisalsogood.)

ToddH20,addDEPCto0.1%

ShaketogettheDEPCdropletsintosolution

Leaveat37deg.overnight

Autoclave20minutestodestroytheDEPC

0.1MsodiumphosphatepH7.0(make1liter)

AdjusttotheappropriatepHbymixing~2/5volumeofmonobasicwith~3/5volumeofdibasic0.1Msodiumphosphate

Makeusingcleantechnique,addDEPCto0.1%

ShaketogettheDEPCdropletsintosolution

Leaveat37deg.overnight

Autoclave20minutestodestroytheDEPC

6Mglyoxal(deionized)

Glyoxalispurchasedasa40%(6M)solution(e.g.fromSigma).GlyoxalreADIlyoxidizesinair.OxidationofthesolutioncanbedetectedbytheloweringofitspHascarboxylicacidsaccumulate.Thesechargedacidscanberemovedbypassagethroughamixed-bedresin(e.g.Bio-RadAG501-X8).Thismustbedonequickly,minimizingexposureoftheglyoxaltoair,oritwilljustreoxidizeasyouwork!

Ourmethodistouseabakedspatulatoloadabout5mlsofresinintoeachof~3smallBio-Raddispo-columns(fromafreshbag).MeasurethepHofthestartingmaterialbyputtingafewdropsonpHpaper;itwillprobablybe<1.Pourabout10mlsofsolutionoverthefirstcolumn,collectingtheeluateinanRNasefree15mlscentrifugetube.Thenpasstheeluateoverthecolumnagain,collectinginasecondtube.Continuebypassagingthesolutiontwiceeachoverthesecondandthirdcolumns,oruntilthepHofthesolutionappearstohavereachedasteadystate.MeasurethepHaftereachcolumnbyputtingafewdropsonpHpaper.Thegoalistoreach~pH5,althoughinpracticeasteadystatemaybereachedataboutpH4.5.QuicklyaliquotthedeionizedsolutionintoRNasefreescrewcaptubes(~50ulaliquots),sealthecapstightly,andfreezeat-80deg..Eachaliquotshouldbeusedonlyonceandthendiscarded.

Loadingbuffer:50%glycerol,10mMsodiumphosphatepH7.0

Useglycerolfromafreshunopenedbottle,andDEPCtreatedwaterand0.1MsodiumphosphatepH7.0.

1.Inscrewcapmicrofugetubesmix:

6Mglyoxal5.4ul

DMSO16ul

0.1MNaphosphatepH7.03ul

RNA(upto10ug)5.4ul

incubateat50deg.for1hour.

SetupatubeortwoofRNAMarkersaswell;thesecanbepurchasedfromPromega(catalog#G3151).Use2ugofmakersperlane.Thesemarkershavesizesof9488,6225,3911,2800,1898,872,562,and363bases.

2.Pourthegel:use1%agarosein10mMNaphosphatepH7.0.Afterheatinginthemicrowave,coolto70deg.,addsolidsodiumiodoacetateto10mM(toinactivateRNAses),coolto50deg.,andpourthegel.

3.Setupthegel:submergethegelinrunningbuffer(10mMNaphosphatepH7.0).Thislowstrengthbufferrequiresrecirculationduringtherun,soputastirbarineachbuffertank,setupaperistalticpumptorecirculatebufferbetweenthetwotanks.Putastirplateundereachbuffertank.

4.ChilltheRNAtubesonice,spinthemdownbrieflyinthemicrofuge.Add4ulloadingbuffertoeachtube,andimmediatelyloadthesamples.Youmaywanttoaddsomeloadingbuffercontainingxylenecyanolandbromophenolbluetoanextralaneonthegeltomonitorprogressoftheelectrophoresis.Anextrablanklaneshouldbeleftbetweenthemarkerlaneandyoursamplelanes.Thatway,itiseasytocutthemarkerlaneofftheblotandstainit.Ifamarkerlaneisleftontheblotduringprobing,beawarethatsomeofthemarkers(9.4,6.2,0.87kb)hybridizewithpUCvectorsequencesthatwilllikelycontaminateyourprobe;thisisallthemorereasontoleaveaspacebetweenthemarkersandyoursamples.

5.Runthegelat3-4V/cm.Waitabout10minutesafterturningonthepowerbeforestartingthestirbarsandperistalticpump;thisallowstheRNAtorunintothegelfirstsothatthebuffercirculationwon"tdisturbitinthewells.Ittakes2.5hourstorunthegelabout11cm;thegelandbufferwillbecomewarmduringtherun.

6.Afterthegelisrun,nofurthertreatmentofthegelisnecessarybeforeblotting.ItisimmediatelyblottedtoNytranin20XSSPE,exactlyasyouwouldforaSouthernblot(seeMichaelKoelle"sSouthernblotprotocol).Afterblottingovernightcarefullymarkthewellsontheblot,UVcrosslinkjustasyouwouldaSouthern,anddrythefilterinavacuumoven(takesabout20minutes).

7.VisualizingtheRNAmarkersand/orsamples:Wefoundthatpost-stainingglyoxalorformaldehydegelswithethidiumbromidedoesn"tworkwell.It"snotpossIBLetorunglyoxalgelsinethidium,sinceglyoxalreactswiththeethidium.IfyoujustwantaquickcheckofwhatanRNAsamplelookslike,itispossibletomixethidiumbromidewithRNAsampleandthenloadthemixtureonaformaldehydegel(seeSambrooketal.forthismethod).Thisgivesagoodstain,butit"snotnearlyasniceaswhatyougetifyoublotthegelandmethylenebluestain,asdescribedbelow.

WhenmakingNorthernblots,however,itispreferabletovisualizetheRNAmarkersdirectlyontheblot,sincethisallowsforthemostaccuratealignmentwiththeautorad.Cutthemarkerlanes(andanyextrasamplelanesyouwanttostain)offtheblot.Soakin5%aceticacidfor15minutes,andthentransferto0.5Msodiumphosphate(pH5.2),0.04%methylenebluefor5-10minutesatroomtemperature.Rinsetheblotinwaterfor5-10minutes,untilthebackgroundiswhite.Themarkersshouldappearassharpbluebandsonawhitebackground.TotalRNAfromC.elegansshouldhavetwoheavyribosomalRNAbandsatabout3.5kband1.7kb,andalightersmearofmRNAcenteredaround2kb.ContaminatingE.colicanbedetectedbythepresenceof3.0and1.5kbE.coliribosomalRNAs.

8.Beforeprobingtheblot,soakitin20mMTrispH8.0at65deg.for30minutes.Thisremovestheglyoxal.

9.ProbethefilterexactlyasyouwouldaSouthernblot.Northernscanbereusedmanytimes,somakesuretoalwayskeeptheblotmoist,andfreezeitinaseal-a-mealbagbetweenuses.It"sbesttojustletthesignalfrompreviousprobingsdecayovertimebeforereusingtheblot;repeatedlystrippingthefiltermaydecreasethesignalonsubsequentprobings.