I.IntroductionMostcurrentmethodsofmeasuringgeneexpressionrelyonaveragingmanycellularresponsesorartificialamplificationstepstoreachadetectablethresholdofsignal.Incontradistinction,insituassayscircumventtheseprocedurestoyielddirectsinglecellexpressioninformation.Fluorescenceinsituhybridization(FISH)isthegoldstandardforlocalizationofnucleicacids(FauthandSpeicher,2001;vanderPloeg,2000).Theintroductionofamino-allylmodifiedbases(Langeretal.,1981)allowedthechemicalsynthesisofmultiply-labeledfluorescentoligomerhybridizationprobes(Feminoetal.,1998;Kislauskisetal.,1993).This,inturnallowedtheapplicationofmulticolor/multi-spectralFISH(Nederlofetal.,1990)tovisualizationofmultipleRNAspeciessimultaneously(Levskyetal.,2002).Withtheintroductionofvisualizationtogeneexpressionassayswebegintounderstandthecomplexityofbehavioratthecelllevel,allowingre-investigationofassumedconsistenciesofcellpopulationswithsingle-cellresolution(Elowitzetal.,2002;Levskyetal.,2002).
II.MaterialsandInstrumentationOligomerprobesweredesignedwithOLIGOsoftwarefromMolecularBiologyInsights,andsynthesizedonanAppliedBiosystemsautomatedDNA/RNAsynthesizer(Model392/394).Solid-supportsynthesiscolumnswerefromAppliedBiosystems(dA,Cat.No.400949;dC,Cat.No.400950;dmf-dG,Cat.No.401184;T,Cat.No.400952).Phosphoramidites(dA,Cat.No.10-1000-10;dC,Cat.No.10-1010-10;dmf-dG,Cat.No.10-1029-10;dT,Cat.No.10-1030-10)andanamino-allylmodifiedbaseforattachmentofester-conjugatedfluorophores(C6-dT,Cat.No.10-1039-05)wereobtainedfromGlenResearch.OligonucleotidePurificationCartridges(OPCTM,Cat.No.400771)and2Mtriethylamineacetate(TEAA,Cat.No.400613)werefromAppliedBiosystemsandtheanhydrousacetonitrile(Cat.No.40-4050-50)wasfromGlenResearch.Trifluoroaceticacid(TFA,Cat.No.BP618-500)wasfromFisherandtriethylamine(TEA,Cat.No.T-0886)wasfromSigma.FluorophoreswerepurchasedfromAmersham(Cy3,Cat.No.PA23001;Cy3.5,Cat.No.PA23501;Cy5,Cat.No.PA25001)andMolecularProbes(OregonGreen488,Cat.No.O-6147;AlexaFluor488,Cat.No.A-10235orA-20191).Sodiumbicarbonateforlabelingbuffer(Cat.No.BP328-1),25-mlpipetsformake-shiftsize-exclusionchromatographycolumns(Cat.No.13-674-41E),andPasteurpipettes(Cat.No.13-678-20D)forfillingthecolumnswerefromFisher.SephadexG-50resin(Cat.No.G-50-150)forpurificationwasfromSigma.Columnswerefashionedbyremovingthecottonfromthetopofoneofthesepipetsandusingaportionofittoplugupthetip.Securethe“column”verticallytoaringstandandcapthetipwitha1.5mlEppendorftubetopreventliquidloss.ThevacuumconcentratorsystemusedwasfromSavant(Speed-Vac),andtheUV-SpectrophotometerusedformeasurementsofprobeconcentrationsandlabelingefficiencieswasfromBeckman(DU640).Glasscoverslips(Cat.No.12-542B),glassslides(Cat.No.12-518-103),12NHCl(Cat.No.A144-212),gelatin(Cat.No.G8-500),Parafilm(Cat.No.13-374-12),forcepsforcoverslipmanipulation(Cat.No.08-953-E)andmagnesiumchloride(Cat.No.BP214-500)werepurchasedfromFisher.20%paraformaldehydeforpreparationoffixative(PFA,Cat.No.15713)andcoplinjarsforwashes(Cat.No.72242-01)werefromElectronMicroscopySciences.10Xphosphatebufferedsaline(PBS,Cat.No.1666789),20Xsodiumchloride/sodiumcitrate(SSC,Cat.No.1666681),purifiedbovineserumalbumin(BSA,Cat.No.711454)andE.colitRNA(Cat.No.109541)werefromRoche.TritonX-100(Cat.No.T-9284),formamide(Cat.No.F-4761),shearedsalmonspermDNA(ssDNA,Cat.No.D-7656),4’,6-diamidino-2-phenylindole(DAPI,Cat.No.D-8417),anddiethylpyrocarbonate(DEPC,Cat.No.D-5758)wereobtainedfromSigma.Theglassplatesusedforhybridization(Cat.No.165-1824)wereobtainedfromBioRad.TheProLongAntifadeKitformountingslides(Cat.No.P-7481)wasfromMolecularProbes.UprightfluorescencemicroscopesfromOlympuswereusedtoimagemultiplespectralsignaturesfromtheFISHspecimens(modelsAX70andBX51)withapiezoelectrictranslatorfromPhysikInstrumente(Cat.No.PZ54E)togeneratethree-dimensionalimagestacks.Alternatively,microscopesfeaturinganinternalharmonicdrivemaybeused(e.g.BX61fromOlympus).Illuminationwasprovidedbya100WMercuryarclamp.MicroscopeswereoutfittedwithOlympusPlanApo60x,1.4NAobjectivesandChromaHiQbandpassfilterstoseparatefluorescencesignals.Althoughothermethodshavebeenintroducedtodiscernmultiplefluorescencesignalsfromchromosomes(Schrocketal.,1996),theyhavenotbeensuccessfullyappliedtodetectionofmRNAtranscriptionsites.InLevsky(2002)weperformedcolorcodingoftranscriptsusingthefollowingfiltersfromChroma:DAPI(Cat.No.31000),FITC(Cat.No.41001),Cy3(Cat.No.SP-102v1),Cy3.5(Cat.No.SP-103v1)andCy5(Cat.No.41008).High-resolution,low-noisefluorescenceimageswerecapturedusingCharge-CoupledDevice(CCD)camerasfromRoperScientific(modelsCH-350(502)andCoolSNAP-HQ).AcquisitionanddatamanipulationwereperformedusingIPLabsoftwarefromScanalytics.Toeasedataprocessingandavoidmanualmanipulationsthatintroducebias,wecodedourownfilteringanddataanalysissoftwareintheJAVAProgrammingLanguageusingtheJavaDevelopmentKitandAdvancedImagingLibraryfromSunMicrosystems.
III.ProceduresA.PreparationofFluorescentOligomerHybridizationProbesThisprocedureisaccordingtoKislauskis(1993).
Solutions
1.Diethylpyrocarbonate-treateddistilledwater(DDW):Tomake1liter,add.5mlDEPCto1literofdistilledwater.ShakeorstiruntilDEPCiswelldistributedandthenautoclave.Prepareenoughofthistouseinallothersolutions.
2.Labelingbuffer(0.1MNa2CO3sodiumcarbonate,pH9.0):Tomake100ml,weigh1.06gNa2CO3andcompleteto100mlwithDDW.AdjustpHto9.0byadding10NNaOHandstoreat4°C.
3.2MTEABstock:Tomake500ml,take138.3ml(101g)TEAandfillto400mlwithDDW.UsedryicetobubbleinCO2untilpHisbelow8.0.Completeto500mlwithDDWandstoreat4°C.*TEAisextremelyhazardoussotakecarewhenhandling.Useglasswareinsteadofplasticwarewhenmeasuringandtransporting.
4.Filtrationcolumnrunningbuffer(10mMTEAB):Tomake1liter,take5mlof2MTEABstocksolutionandcompleteto1literwithDDW.Storeat4°C.
5.Filtrationcolumnrunningmatrix:Tomakeapproximately200ml,pour200mlof10mMTEABintoanErlenmeyerflask.Addapproximately5gofSephadexG-50andswirltoabsorbtheliquid.Suspensionwillsettle.Storeat4°C.Priortouse,applyvacuumpressuretotheflasktodegasthesuspensionforatleast2hoursbeforepouringmatrix.
Steps
- 1.Havingselectedageneofinterest,choosefourtofiveregionsforprobefabrication,each50basesinlength.AdjustsearchparameterswithintheOLIGOsoftwaretoreceivebestpossiblesequencesforgenedetection.Severalconsiderationsforprobedesignare:
- i.SpanningdifferentareasofthemRNAincreaseschancesdetection;intronicregionsshouldbeavoided.
- ii.50%GCcontent(orclosetothis)isoptimal.
- iii.Highlystablehairpinsshouldbeavoided.
- iv.Theremustbeenoughwell-spacedresiduesforsubstitutionofmodifiedbases.Thisdependsonthemodifierused.Wespacedfivemodifiedthymidineresiduesat8ormorebasesapart.
- v.Thesequencesmustnotcross-reactsignificantlywithothermRNAs.UseBLASTtotestthis(seehttp://ncbi.nih.gov/blast).
2.Preparethereversedantisensesequenceofeachdesignedoligo,substitutingthemodifiedbasesappropriately.3.Synthesizetheoligosaccordingtosynthesizerspecificationsata0.2µMscale,specifyingTRITYL-ON.Deprotectthecrudeproductsina65°Cwaterbathforonehr.4.Aliquotthecrudeproductinto200-300µlportionsandsetoneasideforimmediatepurification.Vacuum-drytheremainingaliquots,thenre-suspendeachpelletin1.0ml10mMTEABplus5µlTEA.Storetheseat–80°Cforfutureuse.*Asthealiquotsdry,thesolutionsbecomeincreasinglyacidicandmaycausedetritylationoftheoligos.Toavoidthis,addadropofTEAtoeachtubeperiodicallywhiledryingthem.5.PurifytheremainingaliquotusingtheOPCTMaccordingtorecommendedprocedures(AppliedBiosystems).Vacuum-drythefinalpureproduct,thenre-suspendin50mlDDW.DetermineconcentrationofproductusingODmeasurementsat260nm.6.Prepareaprobemixturewithequalamountsofeacholigotoobtainafinalamountof20µg–either4or5µgofeacholigodependingonhowmanyweresynthesized.Vacuum-drythismixture.7.Resuspendthepelletin10µllabelingbuffer,andaddittothereactionvialcontainingapproximately1.0mgofdye-esterconjugate.Alternatively,oligoscanbelabeledaccordingtomanufacturer’sspecifications(AmershamorMolecularProbes).Vortexandleaveatroomtemperatureovernight.8.Assembleasize-exclusionchromatographycolumnbytransferring10mMTEABviaglassPasteurpipetintotheprepared25mlpipet/columnuntilliquidlevelisaboutathirdofthewayup.AddtheG-50suspensioninthesamemannerand,asthematrixsettles,removethe1.5mltube“cap”toallowthematrixtosettleabovethecotton-plugstop,whilepermittingliquidtopassthrough.AfterthematrixhasfilledthepipetpackitdownwithacontinuousflowofTEABfor10-15min.Thiscanbemosteasilyaccomplishedbyusingasiphoningsystemattachedtothecolumn.9.Oncethematrixhaspacked,removethesiphoningattachmentandallowthebuffertorundowntothelevelofthematrix,takingcarenottoletitrunbelow.Addthe10µlvolumeofprobe/dyemixturetothecolumnandwashthereactionvialwithanadditional200-300µloffreshTEAB.Addthewashtothecolumnandallowittobegintorundownintothematrix.Whenthedyeproducthasbeenabsorbedintothematrix,refillthecolumnwithbufferandreattachthesiphoningsystemtoprovidecontinuousliquidflow.10.Asthelabeledprobemixturerunsdownthecolumnitwillseparateintotwobands.Thefirst,fasterbandwillcontainthedesiredpureproduct.Collectcolumneluatesin1.0mlfractionstoincludethisfirstband.Vacuum-drythesefractions.Re-suspendtheselectedfractionsinDDWtoachieveatotalvolumeof500µl.11.MeasureODofthefinalsampletodeterminefinalconcentrationandlabelingefficiencyfortheproductaccordingtospecificationsofthedyemanufacturers(Amersham,MolecularProbes).Afinalconcentrationof40ng/µlwouldindicatethatall20µgofoligoinitiallylabeledhasbeencollected.12.Labeledprobecanbestoredat4°C,orat–20°Cforlongertermstorage.B.PreparationofCellSamplesSolutions
- 1.Coverslipsin0.5%gelatin:Tomake200ml,sterilizeaboxofcoverslipsbyboilingin0.1NHClfor20min.RinseandwashthecoverslipsinDDWseveraltimes.Weigh1.0gofgelatinandcompleteto200mlDDW.Stirandwarmtodissolvecompletely.Transfersterilizedcoverslipstogelatinsolutionandautoclavefor20min.Storeat4°C.
- 2.10XPBSstock:Tomake500mlofDEPC-treated10XPBS,take500ml10XPBSandadd250µLDEPC.Stirorshaketodissolve;autoclave.
- 3.1MMgCl2stock:Tomake100ml,weigh20.3gMgCl2andcompleteto100mlwithDDW.
- 4.Washingsolution(PBSM):Tomake1liter,take100ml10XPBSstock,add5ml1MMgCl2stockandcompleteto1literwithDDW.
- 5.Extractant(PBST):Tomake1liter,take100ml10XPBSstock,add5mlTritonX-100,andcompleteto1literwithDDW.StirgentlytoallowTritontodissolvecompletely.*ThisextractanthasbeenusedsuccessfullytoremovecytoplasminculturedDLD-1cells.Thestrengthoftheextractantmustbeoptimizedforeachcelltypetoobtainoptimalreductionofcytoplasmicbackgroundwithoutdamagingnucleiorlossofcells.
- 6.Fixative(4%PFA):Tomake50ml,takeone10mlvialof20%paraformaldehydestock,add5ml10XPBSstock,andcompleteto50mlwithDDW.Storeat4°C.
Steps
- 1.Growcellsunderstandardconditionsandseedontogelatinizedcoverslipsinapetridish.Cellsaregrowntoempirically-determinedconfluencesuchthattheyaresparseenoughtofacilitateautomatedseparationofnucleiduringimageprocessing,anddenseenoughtohavesignificantamountsforanalysis.
- 2.Anytreatmentsteps,suchasserumstarvationandstimulation,canbeperformedatthispointbeforefixation.
- 3.Washthecellsbrieflywithice-coldPBSM.
- 4.Extractthecellsfor60secondsinPBSTatroomtemperature.
- 5.Washthecellstwicebrieflywithice-coldPBSM.
- 6.FixthecellswiththePFAfixativesolutionfor20minatroomtemperature.
- 7.Washthecellsagaintwicebrieflywithice-coldPBSM.
- 8.Fixedcoverslipsmaybestoredat4°CinPBSMuntiluse.*Furtherextractionandbackgroundreductioncanbeobtainedforsomecelltypesbystoragein70%ethanolat4°C.Insomecasesthiscancausecellstodetachfromcoverslips.
C.HybridizationThisprocedureismodifiedfromFemino(1998)andLevsky(2002).Solutions
- 1.Washingsolution(PBSM):Tomake1liter,take100ml10XPBSstock,add5ml1MMgCl2stockandcompleteto1literwithDDW.
- 2.Pre/post-hybridizationwash(50%formamide/2XSSC):Tomake500ml,take250mlformamide,add50ml20XSSCstockandcompleteto500mlwithDDW.
- 3.Probecompetitorsolution(ssDNA/tRNA):Tomake100µlof10mg/mltotalconcentrationcompetitor,take50µlof10mg/mlshearedsalmonspermDNAandadd50µl10mg/mlE.colitRNA(preparedfromsolidbyadding10mgto1.0mlDDW).Storeat–20°C.
- 4.Hybridizationbuffer:Tomake100µl,take60µLDDWandadd20µLBSA,and20µL20XSSCstock.Preparefreshandholdonice.*Thisvolumeissufficientfor10hybridizationreactions(10coverslips).
- 5.Low-saltwashsolution(2XSSC):Tomake500ml,take50ml20XSSCstockandcompleteto500mlwithDDW.
- 6.Nuclearstainsolution(DAPI):Tomake1liter,take100ml10XPBSstock,add50µL10mg/mlDAPIstock(preparedfromsolidbyadding10mgto1.0mlDDW)andcompleteto1literwithDDW.ShakeormixtodissolveDAPIcompletelyandstoreat4°C.
- 7.Mountingmedium:Tomake1ml,prepareingredientsofProLongkitaccordingtomanufacturer’sspecifications(MolecularProbes)oruseanequivalentmethod.About25µlofmediumisneededpercoverslip.
Steps
- 1.Hybridizationistestedbeforecolor-codingandmultipletranscriptdetection.Westartedbyusingtwobrightdyes(Cy3andCy5)toshowtranscriptionsites.Afterthis,eachgeneisassignedanarbitrarycolorcodeusingcombinationsofdyesandtestedsingly.Onlyafterresultsarereproducibleismultiplexdetectionperformed.
- 2.Usingforceps,placefixedcoverslipsverticallyinacoplinjar,keepingnoteofwhichsidehasthecellsonit.RehydrateandwashthecellsinPBSMfortenmin.*Allwashesareatroomtemperatureunlessotherwisenoted.
- 3.Equilibratethecellsinpre-hybridizationsolutionfortenmin.
- 4.Aliquotprobemixturesforgene(s)tobedetectedintotubesforeachdifferentcombinationoftargetstobeassayed.*Asastartingconcentration,combine20ngofeachprobeforthe20µltotalfinalreactionvolume.Optimalconcentrationsofthedifferentprobemixturesaredeterminedempiricallybybalancingtheresultantcolorsdetecteduponimagingtranscriptionsites.
- 5.Addcompetitorsolutiontotheprobemixture(s)in100-foldexcess.Vacuum-drythismixture,takingcarenottooverdry.
- 6.Re-suspendthedrypelletin10µlformamideandplacethetubesonaheatingblockat85°Cfor5-10min,thenplaceimmediatelyonice.
- 7.Add10µlofhybridizationbuffertoeachtube,givingafinalreactionvolumeof20µl.
- 8.Wrapaglass/plasticplatewithparafilmtoallowenoughworkingspacefortheamountofreactionsyouhave.Doteach20µlreactionvolumeontotheplate,farenoughapartsuchthatcoverslipscanbeplacedovereachvolumewithoutoverlap.
- 9.Removecoverslipsfrompre-hybridizationsolutionandblotoffexcessliquid.Placeeachcoverslip–cellsidedown–onthehybridizationmixalreadydottedontotheplate.
- 10.Wrapanotherlayerofparafilmovertheplateandcoverslipstosealthereactions.Pressaroundtheedgeswithapenorsimilarinstrument.
- 11.Incubatetheplateat37°Cforthreehr,alongwithasufficientamountofpre-hybridizationsolutiontowashthecoverslipstwiceafterhybridization.
- 12.Removethetoplayerofparafilmandcarefullyliftthelowerlayersothatthecoverslipscanberemovedeasilywithoutexcessivemanipulation.Placethecoverslipsbackintocoplinjarswiththepre-warmedwash–keepingtrackofthecell-side–andincubatefor20minat37°C.Changeandrepeatthiswashforanother20min.
- 13.Changethesolutionwith2XSSCandincubateatroomtemperaturefortenmin.
- 14.ChangethesolutionwithPBSMandincubateatroomtemperaturefortenmin.
- 15.CounterstainthenucleibychangingthesolutionwiththepreparedDAPIandincubatingatroomtemperatureforonemin,thenwashingwithPBSM.
- 16.ChangethePBSMandkeepatroomtemperatureuntilreadytomount.
- 17.Mounteachcoverslip(cell-sidedown)ontoglassslides,usingfreshlypreparedantifademountingmedium.Blotoffexcessliquidandstoreat–20°C.
D.MicroscopyandImageAnalysisTheseproceduresarefromLevsky(2002).
Steps
- 1.Imagestacksareacquiredwithhighindexoilimmersiononafluorescencemicroscopeoutfittedforopticalsectioning.Weusedastepsizeof.5µmtogenerateimagevolumesastranscriptionsitesignalsarebrightanddidnotrequiremorefinelyspacedplanesonoursetup.Forfutureprocessingstepsandfordetectinglessbrightsignals,closeropticalsectionsmaybeneeded.Weusedthe60Xobjectiveandadditionalmagnification(whennecessary)toyielddigitalimagesofroughly100nm-perpixelresolution.ThetotalmagnificationshouldbeadjustedtoyieldsimilarresolutiongiventhephysicalsizeofelementsontheCCDcameraused.Highresolutionenablesmorphometricprocessingofthesignals.
- 2.Imagevolumesfromdifferentfluorescentchannelsarenormalizedbycontrastenhancementtoensureinterpretationisindependentofrelativeintensity.Thiscanbeperformedwithcommercialsoftware,suchasIPLab(Scanalytics),fortheentirethree-dimensionalimagestacksatonetimetoensurethatthesampleisanalyzedevenly.The‘blackvalue’fortheenhancementshouldbesettotheapproximateextra-nuclearnoiselevelforthesample,whichcanvarymarkedly.The‘whitevalue’shouldbeslightlyabovetheintensityforthecenterofthebrightestsignals,namelynuclearsitesoftranscription.
- 3.Digitalsignalenhancementcanbeapproachedbytwomethods–directanalysisofthree-dimensionalimagesorsplittingtheimageintotwo-dimensionalslices,slice-by-sliceprocessing,andfinally,collationofthedataintoathree-dimensionalrepresentation.Bothapproachesrequiresimilarfilteringalgorithms,butcurrently-availableimplementationsgenerallyrequiredecompositionintoslicesastheycanonlyprocesstwo-dimensionalimages.Eitherway,thebasicmethodofsignalenhancementissimpleconvolutionfilteringusingakernelthatapproximatesthesizeofthetargetsignal.Thisimpliesthatthekernelshouldbeadjustedtoapproximatethesizeofempiricallyobservedsitesoftranscription,asdeterminedbythemagnificationusedinimageacquisition.Thedesignedkernelshouldincludesurroundpenaltytodecreasethechancesoffalse-positivedetectionoflargerareasoffluorescencenoise(intrinsicorextrinsictothesample).Thecenter,orpositivelyscoredpartofthekernel,shouldbelargeenoughtoignorespecularnoiseandcameradefects,whichcanappearashighlyintensesinglepixels.
- 4.Positivedetectionofsitesoftranscriptiondependsonempiricselectionofathreshold.Ifcontrastenhancement(step1)wasperformedcorrectly,thisshouldallowasinglecolorleveltobeusedtodistinguishbetweenbackgroundlevelsandtranscriptionsitecolorcodesinallfluorescencechannels.Thisproceduremaybeperformedusingasegmentationalgorithmforeachcolorcombinationusedfordetectionintheimage,suchthatonesinglesoutsitesofeachidentityoneatatime.Findingthistedious,weprefertodetectallsupra-thresholdsignalsanddeterminethecolor-codedidentityatoncebycodingasimplealgorithm.Thisprocedureinvolvesscanningtheimagepixelbypixelforsupra-thresholdsignal,recordingeachputativesignal,andmarkingoffcontiguousregionssurroundingthesignalsuchthattheyarenotscoredmorethanonce.Locationofthesignalandintensityinallcolorbands(bothpoint-wiseandwithsurrounding-area)arerecorded.Theintensityvaluesarecomparedwiththethresholdandidentityofthesiteisassigned.
- 5.Forvisualizationpurposes,apseudo-colored,flattenedtwo-dimensionalrenderingisprepared.Forabackground,weprefertousethemiddleZ-sliceofthenuclearcounter-stainimage.Transcriptionsitelocationsandidentities(nowarbitrarilypseudo-coloredanddepictedwithanartificialMarkerintheimage)areshown.WehaveaddedanumberadjoiningthesitetomarktheZ-sectionfromwhichthecenterofthesitewasdetected.Thisisnecessarysincethefilteredandthreshold-correcteddatacontainsmorethanthreecolorsofimagesandcannotbedepictedunambiguouslyinred-green-bluecolorsystems.
- 6.Nuclearboundsaregeneratedbybinarizationandsimpleflood-fillofthenuclearcounter-stainimage.Binarizationrequiresasinglethresholdtobechosentodistinguishbetweenintra-nuclearandextra-nuclear;thiscanusuallybedonegivenappropriateexposureofthecounter-stain.Flood-fillalgorithmswillonlyworkwithdiscretelyseparatednucleiandmustbemodifiedsignificantlytointerpretoverlappingsignals.Nucleiforwhichtheflood-fill-definedareaextendstotheedgeoftheimageplaneshouldberuledoutforfurtheranalysisastheircontentsareincompletelyimaged.
- 7.Joiningtheresultsofsteps4and5nowyieldsthedataofsinglecellgeneexpressionprofiles–asettranscriptionsitesforeachnucleusanalyzed.Eachtranscriptionsitedetectedinafieldisplacedontotheflood-fillmapandassignedanucleus.Setsofnucleardataareexportedforfurtherstatisticalstudy.
IV.Pitfalls
- 1.Theoverlapoffluorophorecolorsshouldbecarefullyconsideredwhendesigningabarcodingscheme.Considerationofthestrengthoffluorophores,theseparationbetweenemissionspectra,excitationcharacteristicsofthelamp,andthefiltersetstobeusedtodiscernsignalsiscritical.
- 2.WhenassemblingtheG-50columnandloadingsampledonotlettheliquidlevelrunbelowthematrix.Thiswillcreatecracksandbubbles,potentiallydisruptingcompletebandseparationandaddingtocontaminationofproductwithfreedye.
- 3.Poorlylabeledprobes(<40%)canfailtodetecttranscriptionsites.Toincreaselabelingefficiency,multipleseriallabelingsandpurificationscanbeperformed.
- 4.Probemixturesthathaveasuspiciouslyhighleveloflabeling(>80%)maycontainfreedye,whichwillincreasebackground.MultiplepurificationsbyG50columncanbeusedtoremedythis.
- 5.Whenplacingthecoverslipsdownontotheparafilm-coatedplates,careshouldbeexercisedtoavoidbubblesoccurringandtherebypreventingtotalcontactoftheprobewiththecoverslip.Alsotakecarenottotouchormovethecoverslipsexcessivelyoncetheyareplacedontotheparafilmasthismaycontributetocelldetachmentanddamage.
- 6.Somecelltypeshavehighinherentautofluorescenceobscuringnuclearsignals.Carefulprocessingwithadequateextractioncanremedythisattimes.Additionalprocessingstepsmaybenecessaryforrecalcitrantnoiseproblems.
- 7.Transcriptcolorcodesinwhichthecolorsareinadequatelybalancedmay‘decay’suchthattheobservedsignalismisinterpretedasadifferentcolorcodecontainingasubsetoftheoriginalcode.Thisisespeciallyproblematicunderconditionsoflowtranscriptabundanceandwithlessintensefluorophores(liketheFITC-derivatives).Colorcodesmustbecarefullytunedbeforemultiplexdetection.
IV.References
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- Fauth,C.,andM.R.Speicher.2001.Classifyingbycolors:FISH-basedgenomeanalysis.CytogenetCellGenet.93:1-10.
- Femino,A.M.,F.S.Fay,K.Fogarty,andR.H.Singer.1998.VisualizationofsingleRNAtranscriptsinsitu.Science.280:585-90.
- Kislauskis,E.H.,Z.Li,R.H.Singer,andK.L.Taneja.1993.Isoform-specific3"-untranslatedsequencessortalpha-cardiacandbeta-cytoplasmicactinmessengerRNAstodifferentcytoplasmiccompartments.JCellBiol.123:165-72.
- Langer,P.R.,A.A.Waldrop,andD.C.Ward.1981.Enzymaticsynthesisofbiotin-labeledpolynucleotides:novelnucleicacidaffinityprobes.ProcNatlAcadSciUSA.78:6633-7.
- Levsky,J.M.,S.M.Shenoy,R.C.Pezo,andR.H.Singer.2002.Single-cellgeneexpressionprofiling.Science.297:836-40.
- Nederlof,P.M.,S.vanderFlier,J.Wiegant,A.K.Raap,H.J.Tanke,J.S.Ploem,andM.vanderPloeg.1990.Multiplefluorescenceinsituhybridization.Cytometry.11:126-31.
- Schrock,E.,S.duManoir,T.Veldman,B.Schoell,J.Wienberg,M.A.Ferguson-Smith,Y.Ning,D.H.Ledbetter,I.Bar-Am,D.Soenksen,Y.Garini,andT.Ried.1996.Multicolorspectralkaryotypingofhumanchromosomes.Science.273:494-7.
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