ThisprocedurewasfirstdescribedbyBertrandetal(ProceedingsoftheNationalAcademyofScienceUSA(1993)Vol.90pp.3496-3500,andNucleicAcidsResearch(1994)Vol.22pp.293-300)todemonstratethatribozymescouldbeenzymaticallyactiveinvivo.WeadaptedthemethodtoshowthatcertainoligodeoxynucleotidescoulddirecttheactivityofendogenousribonucleaseHtocleavetargetmRNAinintacthumanleukaemiacellsfollowingstreptolysinO-mediateddelivery(method)oftheantisenseeffectors(reference1,reference2).ThetheoryofreverseligationmediatedRT-PCR(RL-PCR)isdescribedelsewhereinthesepages.

RL-PCRcanbebrokendownintoanumberofsimplesteps.SynthesisbyinvitrotranscriptionandpurificationofanRNAlinkerspecies.ExtractionoftotalRNAfromcellsintowhicholigodeoxynucleotidehasbeendeliveredby,forexample,streptolysinOpermeABIlization.LigationoftheRNAlinkertoallavailable5"monophosphatesinthepurifiedtotalcellularRNAsample.ReverseTranscriptionoftheRNAusingagene-specificprimer.AmplificationofspecificfragmentsbyPCRusinglinker-specificandgene-specificprimers.Sub-amplificationofthefirstPCRproductusinglinkerspecificandnested,labelled,genespecificprimers.AlternativelythefirstPCRmaybesequencedusingthenested,labelled,gene-specificprimer.

Reagents/Solutions

• 5µMTemplateforlinkerRNAtranscript(duplexofRLPCRoligonucleotides1and2)in30mMNaCl,2mMTris-HCl,0.2mMEDTApH8.0.
• 5xTranscriptionBuffer.
  • 0.2MTris-HClpH7.5
  • 50mMNaCl
  • 10mMspermidine
  • 30mMMgCl₂
• 0.1Mdithiothreitol
• 1MMgCl₂
• BacteriophageT7RNApolymerase(200U/µl)
• RNaseInhibitor(~40U/µl)
• 100mMNTPs
• 20%Acrylamide-bis(19:1),1xTBEbuffer.
• 4Mguanidinethiocyanate,5mMsodiumcitratepH7,0.5%sarkosyl
  • To50gGuanidinethiocyanateadd
  • 58.6mlH₂O
  • 3.52ml0.75MsodiumcitratepH7.0
  • 5.28ml10%sarkosyl
• 2MsodiumacetatepH4
• 2-mercaptoethanol
• watersaturatedphenolpH4.0-4.3
• Chloroform:isoamylalcohol(49:1byvolume)
• Isopropanol
• T4RNAligase(6U/µl)+10xreactionbuffer(Boehringer).
• RNaseInhibitor(RNaseBlockI40U/µl,Stratagene).
• DNaseI(20-30U/µl)(Boehringer).
• Linearpolyacrylamide(2.5µg/µl,Gaillard&Strauss,NucleicAcidsResearch1990Vol.18pp.378).
• 10mMEDTApH8.0.
• Pfupolymerase(native)+10xreactionbuffer1(Stratagene).
• SuperscriptRTase(Gibco).
• Gene-Specificreversetranscription(RT)primer.
• dNTPs10mMeach.
• RNaseA1ng/µl(boiled)
• RNALinkerspecificprimer(RLPCRoligonucleotide3).
• Nestedgenespecificprimer(GSP).
• Labelled,nested,secondgenespecificprimer(DigP).
• SequencinggelloADIngbuffer(95%formamide,10mMNaOH,0.05%bromophenolblue,0.05%xylenecyanol)
• Sequencinggelmix(6-8%polyacrylamide:bis(19:1),7Murea,1xTBEbuffer)
• SequencingGradeTaq+5xreactionbuffer(Promega).
• d/ddNTPmixes(20µMdATP,dCTP,dTTP,7-deazadGTP:Amix350µMddATP,Cmix200µMddCTP,Gmix30µMddGTP,Tmix600µMddTTP).
• Ethanol,icecold.
• Lightmineraloil.

TranscriptionandpurificationofRNAlinker

1. HybridizeRLPCR1(5"...TTTCAGCGAGGGTCAGCCTATGCCCTATAGTGAGTCGTATTA...3")andRLPCR2(5"...TAATACGACTCACTATAG...3").
   1. Add100µl100µMRLPCR1to100µl100µMRLPCR2and50µl1xTEN(150mMNaCl,10mMTris-HCl,1mMEDTApH8.0)
   2. Heatto70°Cfor5minutes,allowtocooltoroomtemperatureslowly.
   3. Analyseasmallaliquotbynon-denaturingpolyacrylamidegelelectrophoresistoensurethathybridhasbeenformed.
   4. Dilutehybridto5µMbyadditionof1750µl0.2xTEN
2. Transcriptionreaction.(ThesearetheconditionsofMilliganetalNucleicAcidsResearch1987Vol.15pp.8783-8798).PlacethefollowinginanEppendorfinthefollowingorder.
   1. 153µlH₂O
   2. 50µl0.1Mdithiothreitol
   3. 7µl1MMgCl₂
   4. 100µl5xtranscriptionbuffer
   5. 20µl100µMATP
   6. 20µl100µMCTP
   7. 20µl100µMGTP
   8. 20µl100µMUTP
   9. 20µl5µMRLPCR1/RLPCR2hybrid
   10. 20µlRNaseinhibitor(~40U/µl)
   11. 50µlT7RNApolymerase(200U/µl)
3. Incubatetranscriptionreactionfor6hoursat37°C.
4. Fractionatereactiontemplateandproductsbyelectrophoresisthrougha20%non-denaturingpolyacrylamidegel.
5. IdentifythetranscriptbyUV-shaddowingoverathinlayerchromotographyplate.Cutoutthebands.
6. Mashthegelsliceswhichcontainthelinkertranscript.
7. ExtractthelinkerRNAfromthegelbyconstantagitationin~2volumesoflysisbuffer(20ml4MGuCSNbuffer+2ml2MsodiumacetatepH4.0+200µl2-mercaptoethanol)overnight.Centrifuge(10-15,000g,10minutes)topelletthemashedgel.Collectthesupernatent.
8. Re-extractthemashedgelsliceswiththesamevolumeoflysisbufferasabove,butfor2hours.
9. PoolthecollectedsupernatentandprecipitatetheRNAbyadditionof1volumeofisopropanolfollowedbyincubationat-20°Covernight.
10. PellettheprecipitatedRNAlinkerbycentrifugationat10-15,000gfor30minutes.
11. ResUSPendtheRNAin550µllysisbufferandtreatasifpurifiyingtotalRNAfromcells.
12. ResuspendtheresultantRNApelletin100µlH₂Oanddivideinto10x10µlaliquots.
13. Storeat-20°Cuntilrequired.

LigationoflinkerRNAtototalcellularRNA

1. Toeachreactiontubeadd1µgoftotalcellularRNAin4µlddH2O.Storeonice.
2. Makemastermixcontainingthefollowingperreaction:
   • 1µl10xligasebuffer,
   • 1-2µlgelpurified25-merRNA(~500ng)
   • 1µlRNaseinhibitor
   • 0.5µlT4RNAligase(6U/µl)
   • 0.5µlDNaseI(~25U/µl)
   • adjustvolumeto6µl/reactionwithH₂O(1-2µl)
3. Add6µlofmastermixtoeachreactiontube.
4. Incubateovernightat17°C.
5. Incubateat37°Cfor30minutes(ensures~completeDNaseIdigestion).
6. Stopreactionbyadditionof5µl10mMEDTApH8.0and200µlofmodifiedlysisbuffer(lysisbuffer:phenolpH4.31:1byvolume),whichcontains5µglinearpolyacrylamide(co-precipitant)per200µl.Vortexbriefly.
7. Inducephaseseparationbyadditionof25µlchloroform.
8. Recovertheupperaqueusphasetoathermalcyclertubeandprecipitatenucleicacidswith1volume(ca.130µl)propan-2-ol.Storeat-20°Covernight

ReverseTranscription

1. Spindownprecipitatesat10-15,000gfor30minutes.Removeanddiscardsupernatant.Briefsecondspin,pulsetofullspeedthenrelease,discardthe2-4µlofsupernatantsocollected.
2. Resuspendtheprecipitatesin4µlH₂O.Storeoniceuntilreadytoproceed.
3. MakeRTmastermix1withthefollowingcomponentsandwiththisvolumeperreaction
   1. 0.9µlH₂O
   2. 0.7µlPfupolymerasebuffer1
   3. 0.7µl0.1Mdithiothreitol
   4. 0.2µl10µMgene-specificRTprimer
   5. 0.5µlRNaseBlockI
4. HeatRNAsolutionsto95°Cfor5minutesthenplaceimmediatelyat37°C.Incubatefor2-3minutes.
5. Add3µlofRTmastermix1perreaction.
6. Incubateat37°Cfor45minutes.
7. MakeRTmastermix2withthefollowingvolumesperreaction.
   1. 1.4µlH₂O
   2. 0.3µlPfupolymerasebuffer1
   3. 0.3µl0.1Mdithiothreitol
   4. 0.5µl10mMdNTPs(each)
   5. 0.5µlSuperscriptRTase
8. Add3µlRTmastermix2perreaction.
9. Incubateat37°Cfor45minutes.
10. Incubateat95°Cfor10minutes.
11. Add1µlRNaseA.
12. Incubateat37°Cfor20minutes.
13. StoreoniceuntilreadyforPCR1.

FirstPCR

1. MakePCR1mastermixwiththefollowingvolumesperreaction.
   1. 4.8µlH₂O
   2. 1µlPfupolymerasebuffer1
   3. 2µl10µMRLPCR3(linkerspecificprimer,5"...GGGCATAGGCTGACCCTCGCTGAAA...3")
   4. 2µl10µMGSP
   5. 0.2µlPfupolymerase(~0.5U)
2. Add10µlPCR1mastermixtoeachRTreaction.
3. Overlaywith50µllightmineraloil.
4. Thermalcycle:~20cycles.

SecondPCR

1. MakePCR2mastermixwiththefollowingvolumesperreaction.
   1. 9.5µlH₂O
   2. 1.5µlPfupolymerasebuffer1
   3. 1.5µl10µMRLPCR3
   4. 2µl10µMlabelledprimer
   5. 0.3µl10mMdNTPs
   6. 0.2µlPfupolymerase
2. Place15µlPCR2mastermixintofreshthermalcyclertubes.
3. Overlaywith50µllightmineraloil.
4. Add5µlPCR1reaction.
5. Thermalcycle:~5to10cycles.
6. Remove2µlto8µlsequencinggelloadingBuffer.
7. Heatto95°Cfor10minutes.
8. Quenchonice,storeoniceuntilanalysed.
9. Fractionatethrough6-8%polyacrylamidesequencinggel(geltemperature50°C).
10. Standarddetectionprocedureforyourlabeltype.Digoxigeninorfluoresceinlabelledprimerswillrequirethenucleicacidsinthegeltobeelectroblottedordiffusionblottedontonylonmembranepriortochromogenicdetection(method).

RL-PCRSequencing

Pleasenotethisprotocolisanadaptionofthe(Promega)fmolesequencingprocedure.

1. Remove2µlofPCR1to18µlH₂O.
2. Place2µlofthed/ddmixesintoeachoffourtubes(iean"A"tube,a"C"tubeetc.)
3. Sequencingmix:
   1. 8µlH₂O
   2. 5µl5xsequencingreactionbuffer
   3. 2µl1µMlabelledprimer
   4. 1µlofthedilutedPCR1reaction
   5. 5UPromegasequencinggradeTaqpolymerase
4. Add4µlofsequencingmixtod/ddNTPtubes.
5. Overlaywith25µllightmineraloil.
6. Thermalcycle:~30cycles.
7. Add4µlsequencinggelloadbuffer,vortexandcentrifugetomix.Incubateat90°Cfor>5minutes.
8. Loadhotsamplesontostandardsequencinggelwhichhasbeenpre-heatedto50°C.
9. Fractionatebyelectrophoresis.
10. Standarddetectionprocedureforyourlabeltype.

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