Differential Display of RNAs

Thedifferentialdisplayprotocoldescribedhereisbasedontheprincipledescribedby:Liang,P.andA.B.Pardee:DifferentialdisplayofeukaryoticmessengerRNAbymeansofthepolymerasechainreaction,Science257,967-971(1992)Theoligonucleotidesweusearekits,contaning20decamers,fromOperonTechnologiesInc..I.DNaseItreatmentofRNA

• mixandincubatefor30min.at37°C:50µgRNA,10µlRNaseinhibitor(1U/µl),1µlRNase-freeDNaseI(10U/µl),5µl0.1MTris-ClpH8.3,5µl0.5MKCl,5µl15mMMgCl2
• phenolextractonce
• precipitatewith5µlNaAcetateand200µl100%Ethanol
• incubateatleastfor30minat-80°C
• spin,washandredissolvein20µlH2O_DEPC
• measureRNAconcentrationandcheckintegrityonadenaturinggel

II.CDNAsynthesis

• foreachRNAsetupfourreactions(onetubeforeachdegenerateanchoredoligo(dT)primerset-T12MA,T₁₂MC,T₁₂MG,T₁₂MT;whereMisA,CorG)
• diluteDNA-freeRNAto0.1µg/µlwithH₂O_DEPC,keeponice
• setupcDNAsynthesisreactionforeachdegenerateanchoredoligo(dT)primerset:
• 4µl5xreversetranscriptasebuffer,2µlDTT(0.1M),1.6µl4dNTPmix(250µM),200ngRNA,2µlT₁₂MNprimer(10pmol/µl),withH₂Oto19µl
• incubatefor5minat65°Candfor10minat37°C
• add1µlMMLVreversetranscriptase(200U/µl)
• incubatefor50minat37°C
• inactivateMMLVRTbyincubationfor5minat95°C
• useimmediatelyforPCRamplificationorstoreat-20°C

III.PCRreaction

• allPCRsshouldbedoneindoubletsandalwaysruninthesamemachine(tominimizevariations)
• preparemastermixforallPCRreactionswhichcontainthesameT₁₂MNprimer,aliquot18µlintoeachtubeandthenaddthearbitraryprimer(=decamer)
• mixforeach20µlPCRreaction:9.2µlH₂O,2µl10xPCRreactionbuffer,1.6µl4dNTPmix(25µM),2µlT₁₂MNprimer,2µlcDNA,0.2µlTaqDNApolymerase(5U/µl),1µl[a-33P]dCTP
• aliquotandadd2µlarbitraryprimer(2pmol/µl)
• runPCR:40cycles(94°C,30sec.)(40°C,2min.)(72°C,30sec.),1cycle(72°C,5min.)
• storePCRreactionsat-20°Cuntilthegelrun

IV.denaturinggel

• prepare6%denaturingpolyacrylamidegel(7Murea,1xTBE),prerunfor30minat60W
• mix3.5µlofthePCRreactionwith2µlformamideloADIngbuffer,incubate3minat95°C,chillonice
• loadsamples(togetherwithalabelledsizeMarker)andrungeluntilxylenecyanoldyenearlyreachesthebottom(3hours)
• placegelwithoutfixationonWhatman3MMfilterpaperanddryonageldryer
• autoradiographfor24to48hours

V.isolationofPCRfragment

• cutoutbandofinterestwithacleanrazorblade
• soakin100µlH2Ofor10minatroomtemperatureinamicrocentrifugetube
• boilfor15min
• spinandtakesupernatant(containingtheDNA)intofreshtube
• precipitatewith10µl3MNaAcetate,5µlglycogen(10mg/ml),400µl100%Ethanol
• incubateat-70°Cforatleast30min
• spin,washwith85%Ethanolandredissolvein10µlH2O

VI.reamplification

• reamplifyina20µlPCRreaction(usingforeachfragmenttheappropriateT₁₂MN-arbitraryprimercombination)
• mix4µloftheisolatedDNA,2µl10xPCRreactionbuffer,1.6µl4dNTPmix(250pmol/µl),2µlT₁₂MNprimer,2µlarbitraryprimer(decamer),0.2µlTaqDNApolymerase(5U/µl)
• usethePCRconditionsasundersectionIII.
• runPCRproductona1.5%agarosegelandisolatefragmentofexpectedsize
• ifthereisnovisIBLePCRproduct,take1µlofthefirstreamplificationandreamplifyagain
• theisolatedPCRfragmentcanbeuseddirectlyforcyclesequencing,subcloningorasprobefornorthernblots