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Linear amplification of limiting amounts of RNA for gene expression studies188bio精品生物—专注于实验室精品爆款的电商平台 - 蚂蚁淘旗下精选188款生物医学科研用品
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Linear amplification of limiting amounts of RNA for gene expression studies

Expressionprofilingofcellsisavaluabletoolinunderstandinggeneregulatorynetworksandinidentifyinggenesnecessaryfornormaldevelopmentalprocesses.Inthehaematopoieticsystem,cellsurfaceMarkersarewelldefinedallowingtheisolationofcellsfromvariousdevelopmentalstagesusingflowcytometry.Howevergeneexpressionstudiesofsolidtissuesareoftenhamperedbythedifficultyinobtainingahomogenouscellpopulation.Oftenappropriatecellmarkersarenotwelldefinedforgivencellstagesthereforemakinggeneexpressionstudiesdifficult.Inadditionstudiesofrarecellpopulationsmeanthattheamountofmaterialobtainedisinsufficientforanalysis.ToovercometheseproblemswehaveemployedasystemwhereagivencellpopulationisGFP-labeledandthensortedbyflowcytometryinordertoobtainapurehomogenouspopulation.RNAwasthenpreparedfromthismaterialandamplifiedbyalinearamplificationmethodallowinglargescalemicroarraygeneexpressionanalysis.Thisapproachwassuccessfullyappliedtogeneexpressionstudiesofthemidbrain-hindbrainorganizerregionofthemousebutcanbeappliedtomanyothersystemstoisolatecellpopulations.

Usingthemouseasamodelorganism,ourgroupinvestigatedtheroleofthepaireddomaintranscriptionfactorPax2inbraindevelopment(Bouchardetal.,2005).Pax2isrequiredfortheformationoftheisthmicorganizer(IsO)atthemidbrain-hindbrainboundary,whereitinitiatesexpressionoftheIsOsignalFgf8.TogainfurtherinsightintotheroleofPax2inmid-hindbrainpatterning,ourgroupsearchedfornovelPax2-regulatedgenesbyCDNAmicroarrayanalysisofFACS-sortedGFP+mid-hindbraincellsfromwild-typeandPax2-/-E8.5embryoscarryingaPax2GFPBACtransgene(Figure1).Oursortingprotocolyielded5,000to10,000GFP+cellsperembryowhichwasinsufficienttogenerateenoughRNAtohybridizetoamicroarray.ThereforeRNAfromthesortedcellswassubjectedtoalinearamplificationasdescribed(Hoffmannetal.,2003)withsomemodifications.Followingthis,theRNAwasusedinamicroarrayscreenasdescribed(Cheungetal.,1999).OurunbiasedchipapproachidentifiedtheEn2,Brn1,(Pou3f3),Sef,Tapp1andnon-codingNcrmsgenesasgeneticPax2targetsthataretotallydependentonPax2functionfortheirexpressioninthemid-hindbrainregion.Notethatgeneswhichshowedonlya2foldregulationonthemicroarraywereconfirmedasPax2targetgenesbyin-situhybridisation.ThisprovesthattheamplificationprotocolislinearandthatgeneswithlowinductionratiosareindeedPax2-regulatedtargetgenes(Figure2).

Backtotop

PreparationoftotalRNAfromlimitingexvivosortedmaterial

Sorting

Cellsweresortedinto1.5mlmicrocentrifugetubescontaining150µLFACSbuffer.Beforesorting,theinnerwallsofthetubewerecoatedwithbuffertopreventcellsfromstickingtothedrysurface.Aftersorting,asmallpercentageofthesortedcellswereusedforreanalysisbyFACStoverifythepurity.Ifnoreanalysisisnecessary,thecellscanbesorteddirectlyintoTRIreagent(Sigma).

RNApreparation

  1. Centrifugecellsat13,000rpmfor30seconds;
  2. Removethesupernatantleavingabout20µlremaininginthetube;
  3. VortexthetubebrieflytobringthecellsintosUSPensionagain,andwhilevortexing,add500µlTRIreagent;
  4. Leavethetubeatroomtemperaturefor5minutes,andthenadd5mgofcarriertRNA.Add100µlofCHCI3andcentrifugeat13,000rpmfor10minutes;
  5. Transfertheupperphaseintoanew1.5mltube,andadd700mlofisopropanol;
  6. PrecipitatetheRNAbyeitherputtingthetubeat-20°Co/norat-80°Cfor1hourfollowedbycentrifugationat13.000rpmfor30minutes;
  7. Washthepelletwith75%EtOH(preparedwithDEPC-H2O)anddissolveinDEPC-H2O(usuallyRNAfrom50,000-100,000cellsareresuspendedin9ml).

LinearamplificationoftotalRNA

PreparationofdscDNAfromtotalRNA

Firststrandsynthesis(inRNAse-freePCRtubes)

  1. DissolveRNAfromatleast5,000cells(ideally50,000-100,000cells)in9mlDEPC-H2O;
  2. Add1µlPAGE-purifiedoligo-dT-T7-primer(500ng/µl),incubatethemixat70°Cfor10minutes,snap-coolonice;
  3. Preparethefollowingreversetranscriptionmix:4µl5xFirststrandcDNAbuffer,2µl0.1MDTT,1µl10mMdNTPmix,1µlRNAsin,2µlSuperscriptIIRT;
  4. Add10µlmixtotheRNA/primersampleandincubateat42°Cfor1hour.

Secondstrandsynthesis(inRNAse-freePCRtubes)

  1. Preparethefollowingmix:86µlDEPC-H2O,30µl5xsecondstrandbuffer,3µl10mMdNTPmix,8µlDNAPolymeraseI,1µlE.coliDNALigase,2µlRNAseH;
  2. Add130µlmixtothefirststrandcDNA(totalvolume150µl),tapthetubegentlytomix;
  3. Spinbrieflyandincubateat16°Cfor2hours;
  4. Add3µlofT4DNApolymerase;
  5. Incubateat16°Cfor5minutes,putat70°Cfor10minutestostopthereaction.
Clean-upofdouble-strandedcDNA(inRNAse-freemicrocentrifugetubes)
  1. Extractthesamplewith150µlPhenol/Chloroform;
  2. AddtheupperaqueousphasefromtheextractionontoaMicrocon-YM50columnalreadypreloadedwith200µlDEPC-H2O;
  3. Re-extracttheorganicphasewith150µlwater;
  4. Loadtheupperaqueousphasefromthesecondextractionontothecolumn;
  5. Centrifugefor8minutesat9,000rpm,discardflow-through;
  6. Add500µlDEPC-H2Oontothecolumn;
  7. Spinat9,000rpmfor8minutes,discardflow-through;
  8. Placethecolumninvertedintoanewtube,centrifugeat2500rpmfor5minutestorelute;
  9. Bringthevolumeoftheeluateto8µlwithDEPC-H2O,incaseofabiggervolume,speedvacto8µl.

Invitrotranscription(IVT)1(inRNAse-freePCRtubes)

  1. AddtheIVTmixconsistingof4µl5xIVTreactionbuffer,6µl25mMrNTPmixand2µlT7enzymemixtothedscDNA(roomtemperature);
  2. Incubateat37°Cfor4hours.

Clean-up(inRNAse-freemicrocentrifugetubes)

  1. Add130µlDEPC-H2Otobringthetotalvolumeto150µl;
  2. PurifyaRNAusingaQiagenRNeasykitaccordingtothemanufacturer’sprotocol;
  3. ConcentrateaRNAto20µlinaspeedvac.

PreparationofdscDNAfromaRNA

Firststrandsynthesis(inRNAse-freePCRtubes)

  1. UseonlyhalfoftheaRNA,i.e.10µl,forfurthersteps;
  2. Add1,5µlofpd(N)9(1µg/µl,dissolvedin10mMTRIS,pH7.5)totheaRNA;
  3. Incubatethemixat70°Cfor10minutes,snap-coolonice.

Reversetranscription

  1. Preparethefollowingmix:4µl5xFirststrandcDNAbuffer,2µl0.1MDTT,1µl10mMdNTPmix,1µlRNAsin,1µlSuperscriptIIRT;
  2. Add9µlmixtotheaRNA/primersampleandincubateat42°Cfor1hour;
  3. Add3µlofRNAseH(removalofDNA/RNAhybrids);
  4. Incubatefor30minutesat37°C.

Secondstrandsynthesis(inRNAse-freePCRtubes)

  1. Add1.5µlPAGE-purifiedoligo-dT-T7-primer(500ng/µl);
  2. Incubateat95°Cfor5minutes,snap-coolonice,incubateat42°Cfor10minutes;
  3. Addthefollowingmix:92µlDEPC-H2O,15µl10xEco-Polbuffer,4µl10mMdNTPmix,8µlKlenowEnzymeand8µlT4DNAPolymerase;
  4. Incubateat16°Cfor2hours.

Clean-upofdouble-strandedcDNA(inRNAse-freemicrocentrifugetubes)

  1. Extractthesamplewith150µlPhenol/Chloroform;
  2. AddtheupperaqueousphasefromtheextractionontoaMicrocon-YM50columnalreadypreloadedwith200µlDEPC-H2O;
  3. Re-extracttheorganicphasewith150µlwater;
  4. Loadtheupperaqueousphasefromthesecondextractionontothecolumn;
  5. Centrifugefor8minutesat9,000rpm,discardflow-through;
  6. Add500µlDEPC-H2Oontothecolumn;
  7. Spinat9,000rpmfor8minutes,discardflow-through;
  8. Placethecolumninvertedintoanewtube,centrifugedat2500rpmfor5minutesforelution;
  9. Bringthevolumeoftheeluateto8µlbyaddingDEPC-H2O.Iftheeluateismorethan8µlthenspeedvacthesampleto8µl.

Invitrotranscription(IVT)2

  1. AddtheIVTmixconsistingof4µl5xIVTreactionbuffer,6µl25mMrNTPmixand2µlT7enzymemixtothedscDNA(roomtemperature);
  2. Incubateat37°Cfor4hours.

Clean-up(inRNAse-freemicrocentrifugetubes)

  1. Add130µlDEPC-H2Otobringthetotalvolumeto150µl;
  2. PurifyaRNAusingaQiagenRNeasykitaccordingtothemanufacturer’sprotocol(seecomment1);
  3. MeasureaRNAconcentrationonanAgilentBioanalyzer2001accordingtothemanufacturer"sprotocol.

TheaRNAnowcanbeusedformicroarrayhybridizations.Followstandardmicroarrayprotocolsforlabelingandhybridizations.After2roundsofIVT,theamountofaRNAtypicallyvariesbetween60-130µgwhenstartingwith50,000sortedcells.Thisisenoughforatleast20microarrayhybridisations.

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