Overview

Theprotocolisdividedintothreesections:

1. ElectrophoresisofanRNApreparationunderdenaturingconditionsinanagarose-formaldehydegel
2. TransferoftheRNAfromthegeltoanylonornitrocellulosemembranebyupwardcapillarytransfer
3. HybridizationanalysisoftheRNAsequencesofinterestusingalabeledDNAorRNAprobe.

I.Agarose/FormaldehydeGelElectrophoresis

Preparegel:Dissolve0.75gagarosein36mlwaterandcoolto60^oCinawaterbath.Whentheflaskhascooledto60^oC,placeinafumehoodandadd5mlof10xErunningbufferand9mlformaldehyde.Pourthegelandallowittoset.Removethecomb,placethegelinthegeltank,andaddsufficient1xErunningbuffertocovertoadepthof~1mm.

Preparesample:AdjustthevolumeofeachRNAsampleto6µlwithwater,thenadd2.5x6µlfreshlypreparedsampledenaturationmix.Mixbyvortexing,microcentrifugebrieflytocollectliquid,andincubate15minat55^oC.Coolonicefor2min,thenadd2µlloADIngdyemix.

Rungel:Runthegelin1xErunningbufferat100voltfor10min,thenat65voltfor90min

II.TransferofRNAfromGeltoMembrane

Preparegelfortransfer:PlacethegelinanRNase-freedishandrinsewithchangessufficientdeionizedwatertocoverthegelfor4x20min.

TransferRNAfromgeltomembrane:

1. Filltheglassdishwithenough20xSSPE.
2. Cut2piecesofWhatman1MMpaper,placeitontheglassplateandwetitwith20xSSPE.
3. Placethegelonthefilterpaperandsqueezeoutairbubblebyrollingaglasspipet.
4. Cutfourstripsofplasticwrapandplaceovertheedgesofthegel.
5. Cutapieceofnylonmembrane(MSI,Catalog#N00HY320F5)justlargeenoughtocoverthegelandwettedinwater.Placethewettedmembraneonthesurfaceofthegel.Trytoavoidgettingairbubblesunderthemembrane.
6. Floodthesurfaceofthemembranewith20xSSPE.Cut5sheetsofwhatman3MMpapertothesamesizeasmembraneandplaceontopofthemembrane.
7. Putpapertowelsontopofthewhatman3MMpapertoaheightof~6cm,andaddaweighttoholdeverythinginplace.
8. Leaveovernight.

Preparemembraneforhybridization:Removepapertowelsandfilterpapersandrecoverthemembraneandflattenedgel.Markinpencilthepositionofthewellsonthemembraneandensurethattheup-downandback-frontorientationarerecognizable.Rinsethemembranein5xSSPE,thenplaceitonasheetofWhatman3MMpaperandallowtotry.PlaceRNA-side-downonaUVtransIlluminator(254nmwavelength),andirradiateforappropriatelengthoftime.

III.HybridizationAnalysis

PrepareDNAorRNAprobe(>108dpm/µg):

TheprobelabeledwithRidiprimerDNAlabellingsystem(AmershamLIFESCIENCE):

1. DilutetheDNAtobelabelledtoaconcentrationof2.5-25ngin45µlofsterilewater.
2. DenaturetheDNAsamplebyheatingto95-100^oCfor5mininaboilingwaterbath.
3. Centrifugebrieflytobringthecontentstothebottomofthetube,andputonicefor10min.
4. AddthedenaturedDNAtothelabellingmixandreconstitutethemixbygentlyflickingthetubeuntilthebluecolourisevenlydistributed.
5. Add5µlofRedivue[32P]dCTPandmixbygentlypipettingupanddown.Centrifugebrieflytobringthecontentstothebottomofthetube,thenincubateat37^oCfor30min.
6. TheprobeispurifiedusingProbeQuantTMG-50microcolumns(Amershampharmaciabiotech):
7. G-50microcolumnpreparation.ResUSPendtheresininthecolumnbyvortexing,loosenthecapone-fourthturnandsnapoffthebottomclosure.Placethecolumnina1.5mlscrew-capmicrocentrifugetubeforsupport,thenpre-spinthecolumnfor1minat3000rpminanEppendorfmodel5415C.
8. Placethecolumninanew1.5mltubeandslowlyapply50µlofthesampletothetop-centeroftheresin,beingcarfulnottodisturbtheresinbed.Spinthecolumnat3000for2min.Thepurifiedsampleiscollectedinthebottomofthesupporttube.

Hybridization:

9. Pre-hybridization:Wetthemembraneinthe5xSSPEandplaceitRNA-side-upinahybridizationtubeandadd5mlpre-hybridizationsolution,thenplacethetubeinthehybridizationovenandincubatewithrotation6hrat42oCforDNAprobeor60oCforRNAprobe.
10. Hybridization:Double-strandedprobewasdenaturedbyheatinginawaterbathfor10minat100^oC,thentransfertoice.Pipetthedesiredvolumeofprobeintothehybridizationtubeandcontinuetoincubatewithrotationovernightat42^oCforDNAprobeor60^oCforRNAprobe.

Autoradiography:

11. Themembranewaswashedtwicefor5-10minwithwash-bufferatroomtemperature,andtwicefor15minat65^oC.forwithprewarmed(65^oC)wash-buffer.
12. Removefinalwashsolutionandrinsemembranein5xSSPEatroomtemperature.BlotexcessliquidandcoverinUV-transparentplasticwrap.Donotallowmembranetodryoutifitistobereprobed.
13. Blotwasexposedat-80^oCunsingKodakXARfilmandx-rayintensifyingscreens.

ReagentsandSolutions

1. 10xE:0.2MMOPS,0.05MNaAcand0.005MEDTA,adjusttopH7.0withNaOH;
2. Sampledenaturationmix(100µl):64.6µlFormamide,22.6µlFormaldehydeand13µl10xE;
3. Loadingdayemix:50%Glycerol,0.3%Xylenecyanol,0.3%bromophenolblueand1mMEDTA;
4. 20xSSPE:3MNaCl,0.25MNaH2PO4and0.02MEDTA,adjusttopH7.4;
5. Pre-hybridizationsolution:2.5mlformamide,1ml5xP,1.4mlH2O,0.292gNaCland0.1mlspermDNA;
6. 5xP:0.25MTris-HClpH7.5,0.5%Sodiumpyrophosphate,1%Polyvinylpyrolidone,1%Bovineserumalbumine,1%Ficolland5%SDS;
7. Wash-buffer:1%SDS+0.1%20xSSPE.