Sequential RNA and DNA fluorescence in situ hybridization

Anincreasingbodyofevidenceindicatesthatthespatialpositioningofgenesintheinterphasenucleusishighlyrelevantfortheirfunction(Lanctotetal,2007;Meaburn&Misteli,2007;Misteli,2007).Fluorescenceinsituhybridization(FISH)isapowerfultechniquetomapgenelociintheinterphasenucleus.DependingonprotocolFISHcaneitherdetectDNAorRNA.Bothmethodshavelimitations.DNAFISHonlydetectsthephysicallocationofagene,butcannotdetectgeneactivity.RNAFISH,ontheotherhand,detectstranscripts,butmightmissasignificantnumberofalleles,sincenotallallelesofagenearenecessarilytranscribedsimultaneously.ThemostefficientwaytomapgenelociandtheiractivityissequentialRNAandDNAFISH.Thisisanimportanttechniquetouncoverhowgenepositioningislinkedtoactivity.

SimultaneousdetectionofRNAandDNAforagenelocusisnon-trivial.ProceduresduringDNAFISHparticularlydenaturationofcellularDNA,cancausesignificantlossofRNAFISHsignals.Toovercomethisproblem,wehaveemployedtyramidesignalamplification(TSA)todetectRNAFISHsignalsinacombinedDNA/RNAFISHprotocolinwhichRNAisfirstdetectedfollowedbyDNAdetection.Sincetyramidereactswithadjacenttyrosineresiduesinthepresenceofperoxidaseactivityandcovalentlybindstotheresidues,theRNAFISHsignalisprotectedinthesubsequentDNAFISHprocedure.Thisprotocolusesbiotin-labeledsinglestrandedDNAprobesforRNAdetectiondesignedagainstnascenttranscriptsormRNAofthegeneofinterest,andhybridizedprobesarevisualizedusingTSA.AfterRNasetreatment,DNAFISHiscarriedoutwithaconventionalmethod.ThesignalenhancementbyTSAmaygiverisetosomebackgroundbothinthenucleoplasmandremainingcytoplasm,butDNAFISHdifferentiatesthemfromactiveloci.

Withthismethod,wehavesuccessfullymappedactiveandinactiveallelesofIL-4inlymphocytes.ThisenabledustocomparetheradialdistributionofactiveIL-4allelestotheinactiveones,wherewefoundamoreinternalpositioningoftheactiveallelesintheinterphasenucleus(Takizawaetal,2008).

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TheRNAFISHproceduresareessentiallyadaptedfromChakalovaetal,2004.Cellsaregrownonglasscoverslips.

Fixation

1. Fixcellswith4%PFAinPBScontaining10%aceticacidfor15minutesatRTafterremovalofmedia;
2. WashwithPBS3timesfor3minuteseachatRT;
3. ReplacePBSwith70%ethanolandkeepcellsat-20°Cuntiluse(note1);

RNAFISH

1. WashcellswithTNbuffer2timesatRT;
2. ThenincubatecellsinTNbufferfor10minutesatRT;
3. Digestcytoplasmwith0.01%pepsin/0.01NHClfor3minutesat37°C(note2);
4. QuicklyrinsecellswithH₂O(note3);
5. Fixcellswith3.7%PFAinPBSfor5minutes;
6. WashwithPBSatRT3times;
7. Immersecellsintoanethanolseries(70%-90%-100%ethanol,for5minuteseachstep)andairdry;
8. Denatureabiotin-labeledRNAFISHprobefor10minutesat85°Candkeepat37°Cfor30minutes(note4);
9. AddtheRNAFISHprobeasadropontoamicroscopeslide.Placethecoverslipcell-sidedownontopoftheRNAFISHprobe,wipetheexcesssolutionfromthecoverslipandsealusingrubbersolution.Leavethenucleiandprobetohybridizeovernight(O/N)at37°Cinahumidifiedchamber;
10. Washthecellswith2XSSC3timesfor5minuteseachat37°C;
11. PreincubatewithTNTbufferfor5minutesatRT;
12. BlockwithTNTbuffercontaining3%BSAfor20minutesatRT;
13. Inactivateendogenousperoxidaseactivitybyincubationwith1%H₂O₂inPBSfor30minutesatRT(note5);
14. IncubatewithHRP-conjugatedstreptavidindiluted1:100inTNTbuffercontaining3%BSAfor1houratRT(note6);
15. WashwithPBS3times;
16. Incubatewithfluorophore-conjugatedtyramidedilutedin0.0015%H₂O₂at1:100for10minutesatRT(note7);
17. WashwithPBS3times;
18. IncubatewithRNaseA(100µg/mlinPBS)for30minutesat37°C;
19. WashwithPBS3times;
20. Repeatstep7;

DNAFISH

1. Denaturecellsin70%formamide/2XSSCfor10minutesat75°C;
2. Repeatstep7butthe70%ethanolshouldbeice-coldhere;
3. DenatureaDigoxigenin-labeledDNAFISHprobefor10minutesat85°C(note8);
4. Hybridizewiththeprobeovernightat37°C;
5. Washthecellswith50%formamidein2XSSC3timesfor5minuteseachat45°C;
6. Washwith1XSSC3timesfor5minuteseachat60°C;
7. Incubatewith1%H₂O₂inPBSfor30minutesatRT(comment1);
8. Blockwith3%BSA/2XSSC/0.1%Tween20for15minutesatRT;
9. Incubatewithafluorophore-conjugatedanti-DIGantibodydilutedinblockingbufferat1:200for1houratRT;
10. WashwithPBSthreetimesfor5minuteseach;
11. Mountcellswithmountingmediacontaininganti-fadecontainingDAPIsuchasProlongGold.

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1. Itisbettertokeepcellsat-20°CatleastO/Nratherthandirectlygoingtothenextstep.Inourhands,cellscanbestoredforafewweekswithoutsignificantRNAdegeneration.
2. Concentrationofpepsin,temperatureandincubationtimeforappropriatedigestioncanvarybetweencelltypesandneedtobeoptimized.Ideally,thecytoplasmisdigestedbutthenucleusisleftintact.Ifcellsareinpepsintoolongnuclearmorphologybecomesaffected;tooshortanincubationinpepsincanresultinpoorprobepenetration,weaknuclearsignalsandstrongbackgroundintheundigestedcytoplasm.Makesurethesolutionisattherighttemperaturebeforeincubation.Sincepepsinisactivatedanddigestsitself,wewarmup0.01NHClfirstandaddpepsinjustbeforeuse.Foroptimizationexperiments,itiseasiesttocarryoutthedigestiononaphasecontrastmicroscopeandfollowdigestionkineticsinrealtime.
3. Cellscaneasilydetachfromcoverslipsafterpepsindigestion.Solutionsshouldbereplacedgently.
4. Weuse10µlof2ng/µlsinglestrandcDNA(ssDNA)probedissolvedinHybridizationbufferfora12mmroundcoverslip.ssDNAprobeismadebyreversedtranscriptionfrominvitrotranscribedRNAtemplates.Detailsforpreparationoftheprobearein(Chakalovaetal,2004).EitherbiotinordigoxygenincanbeusedasahaptenbutweobtainedbetterresultsusingbiotintolabelRNAFISHprobes.ProbescanbedesignedeitheragainstmRNAorintronsequences.ForthemRNAprobe,weuseplasmidscontainingbacterialpromoterssuchasT7,SP6inthe5’flankingregionofthecDNAsequenceastemplateforinvitrotranscription.Fortheintronprobe,weusePCRforthetargetingsequence(~1000bp)withprimerscontainingabacterialpromotertomakeatemplate.
5. Thisstepcanbeskippeddependingoncelltypes.
6. WeareusingaTSAkitfromInvitrogen(e.g.#T20935).
7. GreenorRedfluorescencecanbeused.
8. Weuse100ngofanick-translatedBACprobefora12mmroundcoverslip.WecommonlypurchaseourBACsfromBACPACResourcesCenter,C.H.O.R.I..Detailsfornick-translationisinourwebsite,http://rex.nci.nih.gov/RESEARCH/basic/lrbge/protocols.html.Thelengthoftheprobesshouldbe200-400bp.Longerprobesgiveincreasedbackgroundsignalsandshorterprobesresultinhomogenous,non-specificstaining.

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Reviewedby:LyubomiraChakalovaandPeterFraser.BabrahamInstitute,Cambridge,UK.

1. Wehadjustonequestionaboutstep7oftheDNAFISH.Wecouldn’tfigureoutwhatthisstepwasforsincetheTSAisalloverbythispoint.[AuthorResponse:WehaveincludedthestepbecausewegotahighbackgroundfortheDNAFISHwithoutit.Wehavenottriedtheprotocolwithoutthisstepsincethen.Perhapsitcouldbeskipped.Usersmighttryboth,withorwithoutthisstep,andempiricallychosethemethodgivingthebestresults.]

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AnexampleofsequentialRNAandDNAFISH.RNAFISHwasperformedagainstIL-4mRNAinmouseThelpertype2cellsandvisualizedwithAlexa594-conjugatedtyramideusingtheTSAtechnique(left).AfterRNasetreatment,DNAFISHwasperformedagainstIL-4usingaDIG-conjugatednick-translatedBACprobe(middle).Nucleiwerecounter-stainedwithDAPI(blue).

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1. ChakalovaL,CarterD,FraserP(2004)RNAfluorescenceinsituhybridizationtaggingandrecoveryofassociatedproteinstoanalyzeinvivochromatininteractions.MethodsEnzymol375:479-493
2. LanctotC,CheutinT,CremerM,CavalliG,CremerT(2007)Dynamicgenomearchitectureinthenuclearspace:regulationofgeneexpressioninthreedimensions.NatRevGenet8(2):104-115
3. MeaburnKJ,MisteliT(2007)Cellbiology:chromosometerritories.Nature445(7126):379-781
4. MisteliT(2007)Beyondthesequence:cellularorganizationofgenomefunction.Cell128(4):787-800
5. TakizawaT,GudlaPR,GuoL,LockettS,MisteliT(2008)Allele-specificnuclearpositioningofthemonoallelicallyexpressedastrocyteMarkerGFAP.GenesDev22(4):489-498