OVERVIEW

FluorescentCDNAmicroarraytechnologyisusefulformakingestimatesoftheabundanceofparticularmessagesrelativetoadesignatedsourceofmRNAthatservesasareferencepoint.CommercialsupportofthistechnologyhasrecentlyreachedalevelwhereitisreasonablefordepartmentsorlargelaboratoriestoconsidersettinguptheirowncDNAarrayfacility.ThissetofprotocolsisintendedtoserveasabasicintroductiontomakingandusingcDNAmicroarraysforthoseembarkingonthispath.TherearethreefundamentaltypesofoperationsrequiredinacDNAmicroarrayexperiment.Thefirstoperation,BASICPROTOCOL1,cDNAAMPLIFICATIONANDPRINTING,dealswithmakingthecDNAmicroarrayitself.ItisnecessarytocollectaninventoryofcDNAbacterialclonesthatrepresentthegeneswhosemessageabundanceyouwishtosurvey.PlasmidtemplatesaremadefromtheseclonesandusedasPCRsubstratestoproduceDNArepresentationsoftheESTinserts.ThePCRproductsarethenpurifiedandspottedontopoly-L-lysinecoatedmicroscopeslides.Inthesecondoperation,BASICPROTOCOL2,RNAEXTRACTIONANDLABELING,RNAisextractedfromthecellsamplestobeexamined,purified,andusedasthesubstrateforreversetranscriptioninthepresenceoffluor-derivatizednucleotides.ThisprocedureprovidesthetaggedrepresentationsofthemRNApoolsofthesamplesthatwillbehybridizedtothegene-specificcDNAdetectorsimmobilizedonthemicroarray.Thethirdfundamentaloperation,BASICPROTOCOL3,HYBRIDIZATIONANDDATAEXTRACTION,coversthestepsinwhichfluor-labeledcDNAshybridizetotheircomplementsonthemicroarray,andtheresultinglocalizedconcentrationsoffluorescentmoleculesaredetectedandquantitated.

FABRICATION

ThisprotocoldescribesthestepsrequiredtoproduceacDNAmicroarray.Gene-specificDNAisproducedbyPCRamplificationofpurifiedtemplateplasmidDNAsfromclonedESTs.ThePCRproductispurifiedbyethanolprecipitation,thoroughlyresUSPendedin3XSSC,andprintedontoapoly-L-lysinecoatedslide.

Materials,Reagents&Solutions

96wellalkalinelysisminiprepkit(EdgeBioSystems,Gaithersburg,MD)LBBroth(Biofluids,Rockville,MD)Superbroth(Biofluids,Rockville,MD).dATP,dCTP,dGTP,dTTP,100mMeach#27-2035-02,storefrozen,-20°C(Pharmacia,Peapack,NJ)PCRprimerAEKM13F(5"-GTTGTAAAACGACGGCCAGTG-3")andAEKM13R(5"-CACACAGGAAACAGCTATG-3")at1mMconcentration,storefrozen,-20°C10XPCRBuffer,#N808-0189,andAmpli-TaqDNApolymerase,#N808-4015storefrozen,-20°C(PerkinElmer,Norwalk,CT)Carbenicillin(Gibco-BRL,Rockville,MD)Ethanol(200ProofUSPEthylAlcohol)1MTris-HCl(pH8)0.5MNaEDTA(pH8)TLowEBuffer20XSSCGlycerol(enzymegrade)SodiumAcetate(tri-hydrate)BoricAcidSodiumHydroxide(1M)GlacialAceticAcidSuccinicanhydride,#23969-0and1-methyl-2-pyrrolidinone,#32863-4(AldrichChemicalCo.,St.Louis,MO)DiethylPyrocarbonate(DEPC)treatedH2OMastersetofclone-purified,sequenceverifiedhumanESTs(e.g.gf211release,ResearchGenetics,Huntsville,AL)96pininoculatingblock(#VP4088,V&PScientific,Inc,SanDiego,CA)AirporeTapeSheets,(#19571,QiagenInc.,Valencia,CA)Sterile96-wellplateseals,(e.g.#SEAL-THN-STR(ElkayProducts,Inc.,Shrewsbury,MA)96-wellU-BottomMicrotiterPlates,#3799and96-wellV-BottomMicrotitrePlates,#3894(CorningInc.,Corning,NY)ThinwallPCRplateandCylclesealPCRplatesealer(e.g.#1038-50-0and#1044-39-4,RobbinsScientificCorp.Sunnyvale,CA)householdone-gallonsealablestoragebags(e.g.GladLock)heatsealablestoragebagsandheatsealer0.2mmSterileFiltrationunitDiamondscribeforwritingonslidesPyrexbakingdish(~24x34x5cm)UVtransparentplasticwrap(e.g.GladClingWrap)30sliderack(stainlesssteel)#113and30slideglasstank,#122(ShandonLipshaw,Pittsburgh,PA)1Lglasstank1Lglassbeaker1LgraduatedcylinderStirbarSlideBox(plasticwithnopaperorcorkliners),(e.g.#60-6306-02,PGCScientific,Gaithersburg,MD)PCRheatcycler(e.g.DNAEngineTetrad,MJResearch,Waltham,MA)Centrifugewithahorizontal("swingingbucket")rotorwithadepthcapacityof6.2cmforspinningmicrotiterplatesandfiltrationplates(e.g.SorvallSuperT21,SorvallInc.,Newtown,CT)37°CShakerincubatorwithholdersfordeep-wellplates37°CWaterbath65°CIncubatorVortexmixerImmunowashmicrotiterplatewasher,#1575(BioRad,Hercules,CA)pHMeterPlatformShakerUVStratalinker2400,(StratageneLaJolla,CA)Stirrer/HotplateRoboticslideprinter-80°CFreezer-20°CFreezer

45%(w/v)SterileGlycerol

450gramsenzymegradeglycerolperliterAutoclaveandstoreatroomtemperature.

TlowEBuffer

1MTris-HCl(pH8.0)10mls0.5MEDTA(pH8.0)0.2mlsDEPCtreatedH2O990mls1000

Autoclaveandstoreatroomtemperature.

Carbenicillinstocksolution

1gramofcarbenicillinin10mlsofsterilewater.

Sterilefilterwitha0.2micronfilter.

Storefrozenat-20°C.

LBwith100µg/mlcarbenicillin

Add1mlofcarbenicillinstocksolutionto1literofLB.

Makefresh.

3MSodiumAcetatepH=6.0

Prepare3Msodiumacetate408.24gramssodiumacetate(tri-hydrate)perliter

Prepare3Maceticacid172.4mlperliter

TitratethepHofthe3MsodiumacetatesolutiontopH6.0withthe3Maceticacidsolution.Filtersterilizeusinga0.2micronfilter.Storeatroomtemperature.

Ethanol/acetatemix

Ethanol(100%)950mlSodiumacetatepH=6.050ml

1000ml3XSSC

DEPCH2O42.5ml20XSSC7.5ml

50ml70%Ethanol

Ethanol(100%)350mlDEPCH2O150ml

500ml

2.1ESTclonegrowth

1.Incubatesealedmasterplatesovernightat37°C.

Mostsuppliersprovidelowdensitybacterialcultures.Replicatingdirectlyfromthesedilutestocksfrequentlyresultsinnon-growthinthesecondaryculture.Ifmakingtemplatefromaplatethathaspreviouslybeenculturedtohighdensitybeforefreezing,thisinitialgrowthstepshouldnotbeused,asitwillreducetheviABIlityofthecultures.

2.Preparesetsofstandard96wellround(U)bottomplatesbylabelingallplatesandplacing100µlofLBbrothcontaining100ug/mlcarbenicillinineachwell.Theseplateswillbeusedasworkingcopies.

Topreservethemastersetofplates,itisusefultomakereplicatecopiesofthemasterplatetoserveasworkingcopieswhenthemasterplateisfirstreplicated.ChecktoinsurethattheESTclonesareinavectorconferringampicillinresistance,asiscommonwithhumanIMAGEclones.,

4.Spinthemasterplatesbriefly,twominutes,at1000rpminahorizontalmicrotiterplaterotortoremovecondensationanddropletsfromthesealsbeforeopening.

Bacterialculturefluidonthesealerscaneasilybetransferredfromonewelloftheplatetoothers,cross-contaminatingthestocks.

5.Partiallyfillacontainerwith100%alcohol.Dipthe96pin-replicatingtoolinthealcohol.Removefromthealcoholbathandthenflamethepins.

6.Allowtheinoculationblocktocoolbriefly,thendipthereplicatingtoolinthemasterplate,andthenintothedaughterplate.Repeatasnecessaryforeachplatethatyouneedtoinoculate.

ItisusefultocolortheplatecornerneartheA-1wellofallmasteranddaughterplateswithaMarkerpenbeforebeginningthereplicationprocess,toreducemistakesinrelativeorientationoftheplates.Thesuggestedplateshaveanotchatthiscorneraswell.

7.PlacetheinoculatedLBplateswithlidsonintoaonegallon"zip-lock"bagcontainingamoistenedpapertowelandgrowovernightat37°C.

Many37°Cincubatorstendtodryoutmicrotiterplatecultures.Placingtheplatesinahighlyhumidifiedbagavoidsthisproblem.

3.Filldeepwellplateswith1mlofSuperbroth(100ug/mlcarbenicillin)perwell.Theseplateswillserveasthesourceofculturefortemplatepreparation.

8.Usingthereplicatingtool,inoculatethedeepwellplatesdirectlyfromthefreshlygrownLBplates.

9.CovertheopeningsofthedeepwellplateswithQiagenAirporeTapeSheetsandplacetheplasticlidoverthesheet.Placetheplatesina37°Cshakerincubatorat200RPMfortwenty-fourhours.

10.Add50µlof45%(w/v)sterileglyceroltoeachwellofanyworkingplatesthataretobefrozen(-80°C)andsubsequentlyusedasculturesources.

2.2Isolateplasmidtemplates

1.Warmthelysisbuffer(EdgeBiosystemsKit)to37°CtodissolvetheSDS.Thisbuffercanbestoredatroomtemperature.

2.AddtheRNAsesolutiontotheresuspensionbuffer(EdgeBiosystemsKit),1ml/100ml.Storeat4°C.Theremainingreagentsfromthekit,neutralizationbufferandprecipitationbuffers,arereadytouseandshouldbestoredat4°C.

3.PreparethereceivingplatesfromtheEdgeBiosystemsKitbyadding350µlofethylalcoholtoeachwellofthereceivingplates.Placethefilterplateontopandsecureinplacewithtape.Handlewithcareasthewellswillbeveryfull.

4.Centrifugethebacterialculturesinthedeepwellplatesat1500xgforsevenminutesinacentrifugeequippedwithahorizontalrotorfor96-wellplates.

5.Invertbrieflyandtapoutexcessmediaonacleanpapertowel.Donotdelayorthepelletswillloosenandmaybelostwhenpouringoffexcessmedia.

6.Resuspendthepelletin100µlofResuspensionBuffer.Vortexuntilentirepelletisre-suspended.Thisstepiscritical.Poorresuspensionofthecellsresultsinclumpsofcellsthatdonotlyseinsubsequentsteps.Thisreducestheyieldanddecreasesthepurityoftheproduct.

7.Add100µlofLysisBuffer.Mixgentlybyrockingtheplatesfromside,avoidshearingthebacterialchromosomalDNA.

8.Add100µlPrecipitationbuffertoeachwell.Mixbriefly.

9.Add100µlNeutralizationbuffertoeachwell.Vortex.

10.Transferthecontentsofthedeepwellstothewaitingfilterplates/receivingplatestacksusingthewideborePipettetipsprovidedinthekits.

11.Centrifugethestackedplatesat1500xgfortwelveminutesinacentrifugeequippedwithahorizontalrotorfor96-wellplates.

12.Removethestackedplatesfromthecentrifuge.Removeanddiscardthefilterplates.Decantthealcoholandfiltratefromthereceiverplate.Touchoffexcessalcoholoncleanpapertowels.

12.Add500µlof70%ethanoltoeachwell.Decantimmediately.Touchoffexcessalcoholoncleanpapertowels.

14.Placeplatesinacleandrawerwithouttheirlids,coverwithacleanpapertowelandallowtodryovernight.

15.Re-suspendDNAin200µlofTLowEBuffer.Sealtopwithplatesealer.Rehydrateat4°Cforatleasttwodaysbeforeusing.Storeat-20°C.

2.3AmplifyESTinserts

1.Foreach96wellplatetobeamplified,prepareaPCRreactionmixturecontainingthefollowingingredients:

2.Label96-wellPCRplatesandaliquot100µlofPCRreactionmixtoeachwell.Gentlytapplatestoinsurethatnoairbubblesaretrappedatthebottomofthewells.

3.Add1µlofpurifiedESTplasmidtemplatetoeachwell.

MarkthedonorandrecipientplatesatthecornerneartheA1welltofacilitatecorrectorientationduringtransferofthetemplate.ItisimportanttowatchthatthepipettetipsareallsubmergedinthePCRreactionmixwhendeliveringthetemplate.Missingtheliquidiseasierwhenmulti-channelpipettesareused.

4.Performthefollowingthermalcycleseries:1initialcycleofheatingto96°Candholdingfor30sec;25cyclesofdenaturingat94°Cfor30sec,reannealingat55°Cfor30sec,andextendingat72°Cfor150sec;onefinalcycleofholdingat72°Cfor5minutes,thencoolingtoambienttemperature.

AfterPCR,platesmadebeheldat4°Cwhilequalitycontrolsareperformed.

2.4CheckPCRproductsbyagarosegelelectrophoresisofESTs

IfthisisthefirsttimethetemplatefortheseESTsisbeingamplified,analyze2µlofeachPCRproductona2%agarosegel.Ifamplifiedproductsfromthistemplatehavebeenpreviouslytested,thenanalyzeonerowofwellsfromeachplateamplified.Gelimagingallowsaroughquantitationofproductwhilegivinganexcellentcharacterizationoftheproduct.Bandsize,aswellasthenumberofbandsobservedinthePCRproducts,contributetounderstandingthefinalresultsofthehybridization.TheuseofgelwellformatssuitableforloADIngfrom96wellplatesandprogrammablepipettersmakesthisformofanalysisfeasIBLeonalargescale.

Materials,ReagentsandSolutions

Electrophoresisapparatuswithcapacityforfour50wellcombs,(e.g.#D3,OwlScientific,Woburn,MA)50XTris-AcetateElectrophoresisBufferMAgaroseDyeSolution(XyleneCyanol/BromophenolBlue)(e.g.#351-081-030,QualityBIOLOGicalInc.,GaithersburgMD)Glycerol(enzymegrade)EthidiumBromidesolution(10mg/ml)100base-pairladdersizestandardProgrammable,12-channelpipetter(e.g.#2019,MatrixTechnologies,Lowell,MA)Disposablemicrotitermixingtrays(e.g.Falcon#353911,BectonDickinson,FranklinLake,NJ)Electrophoresispowersupply

1XTAEBuffer

50XTAEBuffer40mlEthidiumBromide(10mg/ml)0.1mlWater960ml

1000mlLoadingBuffer

Glycerol(enzymegrade)4.0mlDEPCWater0.9mlDyeSolution*0.1ml5.0ml

(*Thissolutionis0.25%(w/v)XyleneCyanoland0.25%(w/v)BromophenolBlue)

100bpSizeStandards

DNAladder(1mg/ml)50µl1MTris-HCl(pH8.0)5µl0.5MEDTA(pH8.0)5µlLoadingBuffer440µl500µl

Method

1.Casta2%agarosegel(1XTAE)withfourcombs(50tooth)andsubmergeinanelectrophoresisapparatuswithsufficient1XTAEbuffertojustcoverthesurfaceofthegel.

2.PrepareareservoirofLoadingBuffer,using12wellsofamicrotiterplate.

2.Programpipettertosequentiallycarryoutthefollowingsteps:

fillwith2µlfillwith1µlfillwith2µlmixavolumeof5µlfivetimesexpel5µl

3.Place12disposabletipsonthepipetter.

4.Load2µlofPCRproductfromwellsA1-A12ofthePCRplate.

5.Load1µlofair.

6.Load2µlofLoadingBufferfromthereservoir.

7.Placetipsincleanwellsofdisposablemixingtrayandallowpipettetomixthesampleandloadingdye.

8.Placethepipettetipina50wellrowsothatthetipcontainingthePCRproductfromwellA1isinthesecondwelloftherow,andtheothertipsareineveryothersucceedingwell.

9.Repeattheprocess(changingtipseachtime),loadingPCRplaterowBstartinginthe3rdwell,interleavedwiththeArow,theCrowstartingatwell26,andtheDrowatwell27,interleavedwiththeCrow.

10.Place5µlof100bpSizeStandards,inwells1and50.

11.Repeatthisprocess,loadingsamplesfromrowsE,F,G,andHinthesecond,50wellrowofgelwells,loadingsamplesfromtwo96wellPCRplatespergel,orsinglerowsamplesfrom16PCRplates.

Toreducediffusionandmixing,applyvoltagetothegelforaminutebetweenloadingeachwellstrip.ThiswillcausetheDNAtoenterthegel,andreducebandspreadingandsampleloss.

12.Applyvoltagetothegelandrununtilthebromophenolblue(fasterband)hasnearlymigratedtothenextsetofwells.

Foragelthatis14cmintherunningdimension,and3cmbetweeneachrowofwells,apply200voltsfor15minutes.

13.Takedigitalphotoofgelandstoreimageforfuturereference.

Thegelsshouldshowbandsoffairlyuniformbrightnessdistributedinsizebetween600to2000base-pairsasinFigure2.Furthercomputeranalysisofsuchimagescanbecarriedoutwithimageanalysispackagestoprovidealistofthenumberandsizeofbands.IdeallythisinformationcanbemadeavailableduringanalysisofthedatafromhybridizationsinvolvingthesePCRproducts.

2.5PurifyPCRproducts

1.Fill96wellV-bottomplateswith200ulperwellofethanol/acetatemix.

Theethanolacetatesolutionusedforprecipitationislessacidic(pH6)thanistypicallyused.Inthisinstance,moreacidicsolutionsproduceprecipitateswhicharehardertoresuspendwithoutimprovingyield.

2.Transfer100ulperwellofPCRproductintoV-bottomplatesandmixbypipettingavolumeof75µlperwellfourtimes.

3.Placetheplatesin-80°Cfreezerforonehourorstoreovernightat-20°C.

Placeplatesat-20°Ciftheyaretobeleftformorethanonehour,aggressiveprecipitationproducesprecipitateswhicharehardtoresuspend.

4.Thawtheplatestoreducebrittlenessandmeltanyice,whichmayhaveformedinthewells.

5.Loadtheplatesintoacentrifugewithahorizontalmicrotiterplaterotorandspinat2600xgfor40minutesat4°C.

6.AspiratethesupernatantfromeachwellusingtheImmunowashplatewasher.

Settingsforthedepthofaspirationbytheplatewasherwillneedtobeadjustedtosuitthemicrotiterplatesused.Itisadvisabletoleaveapproximately10-20mlinthebottomofthewelltoavoiddisturbingthepellet.

7.Deliver200µlof70%ethanoltoeachwellintheplateusingtheImmunowashplatewasher.

8.Centrifugeplatesat2600xgfor40minutes.

9.AspiratethesupernatantfromeachwellusingtheImmunowashplatewasher.

10.Allowtheplatestodryovernightinacloseddrawer.

Donotdryinaspeed-vac.DesiccatedPCRproductsarehardtoresuspend.

2.6ResuspendthePCRproducts

1.Add40µlof3XSSCperwell.Sealplateswithafoilsealer,takingcaretoachieveatightsealovereachwell.

2.Placetheplatesinheatsealablebagswithpapertowelsmoistenedwith3XSSCandsealthebagwithaheatsealer.

Thehighexternalhumiditywithinthesealedbaghelpskeepthevolumesintheindividualwellsfromvarying.

3.Placethebagsina65°Cincubatorfor2hours,thenturnofftheheatintheincubator.

Allowingtheplatestocooldowngraduallyintheincubatoravoidscondensationonthesealers.

2.7CheckPCRresuspensionforyieldbyfluorometricdeterminationofDNAconcentration

Analyze1µlofresuspendedPCRproductfromonerowofwellsfromeachplateona2%agarosegelaspreviouslydescribed.Adequateprecipitationandresuspensionwillproduceveryintensebands,withnomaterialfailingtoleavetheloadingwell,andnosmearofmaterialfromthebandtowardstheloadingwell.

WhileitwouldbeidealtobeabletoexactinglyquantifyeachESTPCRproductandspoteachDNAspeciesatequivalentconcentrations,itisimpracticalformostlabstodosowhenthousandsofESTsmustbeprepared.Fortunately,itispossibletouseastrategywhereexcessDNAisspotted,sothattheexactquantitiesuseddonotproducemuchvariationintheobservedresults.Whenusingthisstrategy,itisnecessarytotracktheaverageproductivityofthePCRreactions.Fluorometryprovidesasimplewaytoobtainanapproximateconcentrationofthedouble-strandedPCRproductinthePCRreactionmix.

Storetheplatesat-20°Cafterresuspension.

Materials,ReagentsandSolutions

Referencedouble-strandedDNA(0.5mg/ml)(e.g.#15612-013Gibco/BRL,Bethesda,MD)96wellplatesforfluorescentdetection(e.g.#7105,Dynex,Chantilly,VA)Fluorometer(e.g.#LS50B,PerkinElmer,Norwalk,CT)FluoReporterBluedsDNAQuantitationKit(#F-2962,Molecularprobes,Eugene,OR)TE12channelmulti-pipettorsComputerequippedwithMicrosoftExcelsoftware

Ds-DNAStandards

50µg/ml100µg/ml250µg/ml500µg/mlµlTE9080500µlds-DNA(0.5mg/ml)102050100

Itisgoodpracticetocheckboththeintegrity(agarosegel)andtheconcentration(absorbance)ofthestandardbeforeuse.

FluorBuffer

Hoechst33258solution*(fromkit)25µlTNEBuffer**(fromkit)10ml

*Hoechst33258solutioncontainsthedyeatanunspecifiedconcentrationina1:4mixtureofDMSO:H2O**TNEBufferis10mMTris-HCl(pH7.4),2MNaCl,1mMEDTA

Quantitatingds-DNA

1.Label96wellplatesforfluorescenceassay.

2.Add200µlofFluorBuffertoeachwell.

3.Add1µlofPCRproductfromeachwellinarowofaPCRplatetoarowofthefluorometryplate.SamplescanbeaddedtorowsAthroughGofthefluorometryplate.

4.Inthefinalrowofthefluorometryplateadd1µlofeachoftheseriesofds-DNAstandards0µg/ml(TEonly),50,100,250and500µg/mlds-DNA.Repeatthisseriestwiceinthefinalrow.

5.Setthefluorometerforexcitationat346nmandemissionat460nm.Adjustasnecessarytoreadtheplate.

6.Ifthefluorometerdoesnotsupportautomatedanalysis,exportthedatatabletoExcel.

7.Testtoseethattheresponseforthestandardsislinearandreproduciblefromtherangeof0to500µg/mlofds-DNA.

8.Calculatetheconcentrationofds-DNAinthePCRreactionsusingthefollowingequationaftersubtractingtheaverage0µg/mlvaluefromallothersampleandcontrolvalues:

[ds-DNA(µg/ml)]=((PCRsamplevalue)/(average100µg/mlvalue))*100

ConstantlytrackingtheyieldsofthePCRsmakesitpossibletorapidlydetectmanyofwaysinwhichPCRcanfailorperformpoorly.ThisassaycanalsobeappliedafterprecipitationandresuspensionofthePCRproductstomonitoroverallrecoveryofproduct.

31.Analyze1µlofamplifiedproductsfromonerowofwellsfromeachamplifiedplatebyfluorometry(SupplementaryProtocol2).

2.8SLIDECOATING

Slidescoatedwithpoly-L-lysinehaveasurfacethatisbothhydrophobicandpositivelycharged.Thehydrophobiccharacterofthesurfaceminimizesspreadingoftheprintedspots,andthechargeappearstohelppositiontheDNAonthesurfaceinawaythatmakescross-linkingmoreefficient.

Materials,ReagentsandSolutions

GoldSealMicroscopeSlides(#3011,BectonDickinson,FranklinLake,NJ)Ethanol(100%)Poly-L-lysine(#P8920,Sigma,St.Louis,MO.)50SlideStainlessSteelRack,#900401,and50SlideGlassTank,#900401,(WheatonScienceProducts,Millville,NJ)SodiumHydroxideStirPlateStirBarPlatformShaker30SlideRack,#196,plastic,and30slideBox,#195,plastic,(ShandonLipshaw,Pittsburgh,PA)SodiumChloridePotassiumChlorideSodiumPhosphateDibasicHeptahydratePotassiumPhosphateMonobasicAutoclave0.2mmFilter:NalgeneCentrifuge:SorvallSuper20SlideBox(plasticwithnopaperorcorkliners),(e.g.#60-6306-02,PGCScientific,Gaithersburg,MD)1LGlassBeaker1LGraduatedCylinder

1MSodiumBorate(pH8.0)

Dissolve61.83gofBoricacidin900mlofDEPCH2O.AdjustthepHto8.0with1NNaOH.Bringvolumeuptooneliter.Sterilizewitha0.2micronfilterandstoreatroomtemperature.

CleaningSolution

H2O400mlEthanol600mlNaOH100g

DissolveNaOHinH2O.Addethanolandstiruntilthesolutionclears.Ifthesolutiondoesnotclear,addH2Ountilitdoes.

Poly-L-lysineSolution

poly-L-lysine(0.1%w/v)35mlPBS35mlH2O280ml350ml

Method

1.Placeslidesinto50slideracksandplaceracksinglasstankswith500mlofcleaningsolution.GoldSealSlidesarehighlyrecommended,astheyhavebeenfoundtohaveconsistentlylowlevelsofautofluorescence.

Itisimportanttowearpowderfreegloveswhenhandlingtheslides.Changeglovesfrequently,asrandomcontactwithskinandsurfacestransfersgreasetothegloves.

2.Placetanksonplatformshakerfortwohoursat60rpm.

3.PouroutcleaningsolutionandwashinH2Oforthreeminutes.Repeatwashfourtimes.

4.Transferslidesto30slideplasticracksandplaceintosmallplasticboxesforcoating.

5.Submergeslidesin200mlpoly-L-lysinesolutionperbox.

6.Placeslideboxesonplatformshakerforonehourat60rpm.

7.RinseslidesthreetimeswithH2O.

8.SubmergeslidesinH2Oforoneminute.

9.Spinslidesincentrifugefortwominutesat400xganddryslideboxesusedforcoating.

10.Placeslidesbackintoslideboxusedforcoatingandletstandovernightbeforetransferringtonewslideboxforstorage.

Thisallowsthecoatingtodrybeforehandling.

11.Allowslidestoagefortwoweeksonthebench,inanewslidebox,beforeprintingonthem.Thecoatingdriesslowly,becomingmorehydrophobicwithtime.

Slideboxesusedforlongtermstorageshouldbeplasticandfreeofcorklining.Theglueusedtoaffixthecorkwillleachoutovertimeandgiveslidesstoredinthesetypesofboxesagreasyfilmthathasahighdegreeofautofluorescence.CleanallglasswareandracksusedforslidecleaningandcoatingwithhighlypurifiedH2Oonly.Donotusedetergent.

2.9.SLIDEBLOCKING

Attheendoftheprint,slidesareremovedfromtheprinter,labeledwiththeprintidentifierandtheslidenumberbywritingontheedgeoftheslidewithadiamondscribeandplacedinadustfreeslideboxtoageforoneweek.

Itisusefultoetchaline,whichoutlinestheprintedareaoftheslide,ontothefirstslide.Thisservesasaguidetolocatetheareaaftertheslideshavebeenprocessed,andthesaltspotsarewashedoff.

1.Placeslides,printedfaceup,incasSEROledishandcoverwithclingwrap.Exposeslidestoa450mJdoseofultravioletirradiationintheStratalinker.

Slidesshouldhavebeenagedatambienttemperatureinaclosedslideboxforoneweekpriortoblocking.

2.Transferslidestoa30slidestainlesssteelrackandplacerackintoasmallglasstank.

3.Dissolve6.0gsuccinicanhydridein325ml1-methyl-2-pyrrolidinoneinaglassbeakerbystirringwithastirbar.

Nitrileglovesshouldbewornandworkcarriedoutinachemicalfumehoodwhilehandling1-methyl-2-pyrrolidinone(ateratogen).

4.Add25ml1Msodiumboratebuffer(pH8.0)tothebeaker.Allowthesolutiontomixforafewseconds,thenpourrapidlyintoglasstankwithslides.

Succinicanhydridehydrolyzesquiterapidlyoncetheaqueousbuffersolutionisadded.Toobtainquantitativepassivationofthepoly-L-lysinecoating,itiscriticalthatthereactivesolutionbebroughtincontactwiththeslidesasquicklyaspossible.

5.Placetheglasstankonaplatformshakerinafumehoodfor20minutes.

Smallparticulatesresultingfromprecipitationofreactionproductswillbevisibleinthefluid.

6.Whiletheslidesareincubatingontheshaker,prepareaboilingH2ObathtodenaturetheDNAontheslides.

7.Aftertheslideshaveincubatedfor20minutes,transferthemintotheboilingH2Obath.Immediatelyturnofftheheatingelementaftersubmergingtheslidesinthebath.AllowslidestostandintheH2Obathfor2minutes.

8.Transfertheslidesintoaglasstankfilledwith100%ethanolandincubatefor4minutes.

9.Removetheslidesandcentrifugeat400rpmfor3minutesinahorizontalmicrotiterplaterotortodrytheslides.

10.Transferslidestoaclean,dustfreeslideboxandletstandovernightbeforehybridizing.

2.10.PRINTING

ThevarietyofprintersandpensfortransferringPCRproductsfromtiterplatestoslidesprecludeshighlydetaileddescriptionsoftheprocess.Thefollowingstepsprovideageneraldescriptionoftheprocessing.

1.Pre-cleantheprintpensaccordingtothemanufacturer"sspecification.

2.Loadtheprinterslidedeckwithpoly-L-lysinecoatedslides(SupplementalProtocol3).

3.ThawtheplatescontainingthepurifiedESTPCRproductsandcentrifugebriefly,twominutes,at1000rpminahorizontalmicrotiterplaterotortoremovecondensationanddropletsfromthesealsbeforeopening.

4.Transfer5to10µlofthepurifiedESTPCRproductstoaplatethatwillserveasthesourceofsolutionfortheprinter.

Printingwithquill-typepensusuallyrequiresthatthevolumeoffluidintheprintsourceissufficientlylow,thatwhenthepenisloweredtothebottomofthewell,itissubmergedinthesolutiontoadepthoflessthanamillimeter.Thiskeepsthepenfromcarryingalargeamountoffluidontheoutsideofthepenshaftandproducingvariable,largespotsonthefirstfewslidesprinted.

5.Runarepetitivetestprintonthefirstslide.Inthisoperation,thepensareloadedwiththeDNAsolution,andthenthepensseriallydepositthissolutiononthefirstslideinthespottingpatternspecifiedfortheprint.

Thistestisruntocheckthesizeandshapeofthespecifiedspottingpattern,anditsplacementontheslide.Italsoservestoverifythatthepensareloadingandspotting,andthatasingleloadingwillproduceasmanyspotsasarerequiredtodelivermaterialtoeveryslideintheprinter.

6.Ifoneormoreofthepensisnotperformingatthedesiredlevel,re-cleanorsubstituteanotherpenandtestagain.

7.Ifallpensareperforming,carryoutthefullprint.

RNAEXTRACTION

ThisprotocoldetailsthemethodsusedtoextractRNAfromcells,purifytheRNAbyacombinationofphaseextractionandchromatography,andpreparealabeledcDNAcopyofthemessagefractionofthepurifiedRNA.TheprotocolalsodescribestheprocessofmakingfluorescentcDNArepresentationsofthemessagepoolswithintheisolatedtotalRNApools.ThisisaccomplishedbyusingthepuretotalRNAasasubstrateforreversetranscriptioninthepresenceofnucleotidesderivatizedwitheitheraCy3oraCy5fluorescenttag.

Materials

TrizolReagent(#15596-018,LifeTechnologies,Rockville,MD)RNeasyMaxiKit(#75162,Qiagen,Valencia,CA)ChloroformEthanol(200ProofUSPEthylAlcohol)DPBS(Dulbecco"sphosphatebufferedsaline)3Msodiumacetate(pH5.2)dATP,dCTP,dGTP,dTTP,100mMeach,storefrozen,-20°C(#27-2035-02,Pharmacia,Peapack,NJ)pd(T)12-18resuspendat1mg/ml,andstorefrozen-20°C(#27-7858,AmershamPharmaciaBiotech)Anchoredoligoprimer(anchored;5"-TTTTTTTTTTTTTTTTTTTTVN-3")resuspendat2mg/ml,storefrozen-20°C(e.g.#3597-006,Genosys)CyTM3-dUTP,1mM,andCyTM5-dUTP,1mM,store-20°C,lightsensitiveRNasinâRnaseinhibitor,store-20°C(#N211A,Promega)SUPERSCRIPTTMIIRnaseH`ReverseTranscriptaseKit,store-20°C,(#18064-014,LifeTechnologies,Rockville,MD)C0t-1DNA,1mg/ml,storefrozen-20°C(#15279-011,LifeTechnologies,Rockville,MD)0.5MEDTA(pH8.0)1NNaOH1MTRIS-HCL(pH7.5)TEpH7.4DEPCwater50XTrisAcetateBuffer15mlroundbottompolypropylenecentrifugetubes50mlconicalpolypropylenecentrifugetubes1.5mlEppendorftubes0.2mlthinwallPCRtubeMicroCon100(AmiconCatNo.42412)Highspeedcentrifugefor15mltubesClinicalcentrifugewithhorizontalrotorfor50mlconicaltubesTissuehomogenizer(e.g.PolytronPT1200withPolytron-Aggregate-Dispergier-und-Mischtechnik147aCh6014#027-30-520-0,BrinkmannInstrumentsInc.,Westbury,NY)

ReagentsandSolutions

RPEBuffer

Add4volumesofethanolpervolumeofRPEconcentratesuppliedinQuiagenKit.

RW1Buffer

SuppliedinQiagenKit

75%EtOH

Ethanol(100%)375mlDEPCH2O125ml

500ml

10xlowTdNTPMix

5xFirstStrandBuffer

ProvidedwithSuperscriptII

TAEBuffer

50XTrisAcetateElectrophoresisBuffer20mlDEPCH2O980ml

1000ml

Method

1.Ifstartingwithcellsharvestedfromtissueculture,washthecellpellettwiceinDPBS.

2.Ifstartingwithcellsfromtissueculture,add1mlofTrizolper2x107cellsandmixbyshaking.Ifstartingwithtissue,add100mgoffrozentissuedirectlyto4mlofTrizol,anddissociatebyhomogenizationwitharotatingbladetissuehomogenizer.

3.Add2/10volumeofchloroformandshakefor15seconds.

4.Letstandfor3minutes.Centrifugeat12,000xgfor15minutesat4°C.

5.Takeoffthesupernatantandaddittoapolypropylenetube,recordingthevolumeofthesupernatant.

6.Add0.53volumesofethanoltothesupernatantslowlywhilevortexing,thiswillproduceafinalethanolconcentrationof35%.

Theethanolshouldbeaddeddropbydropandallowedtomixcompletelywiththesupernatantbeforemoreethanolisadded.Ifahighlocalconcentrationofethanolisproduced,theRNAinthatvicinitywillprecipitate.

7.Addthesupernatantfromanextractionof2x107to1x108cellstoanRNeasymaxicolumn,whichisseatedina50mlcentrifugetube.

8.Centrifugeat2880xginaclinicalcentrifugewithahorizontalrotoratroomtemperaturefor5minutes.

9.Pourtheflow-throughbackontothetopofthecolumnandcentrifugeagain.

AsignificantamountofRNAisnotcapturedbythecolumnmatrixinthefirstpassoftheRNAcontainingsolutionthroughthecolumn.

10.Discardtheflow-throughandadd15mlofRW1buffertothecolumn.

11.Centrifugeat2880xgfor5minutes.

12.Discardflow-throughthenadd10mlofRPEbuffer.

13.Centrifugeat2880xgfor5minutes.

14.Discardflow-throughandaddanother10mlofRPEbuffer.

15.Centrifugeat2880xgfor10minutes.

16.Putthecolumninafresh50mltubeandadd1mlofDEPCtreatedwaterfromthekittothecolumn.

17.Letstandfor1minute.

18.Centrifugeat2880xgfor5minutes.

19.Addanother1mlofwatertothecolumn.

20.Letstandfor1minute.

21.Centrifugeat2880xgfor10minutes.

22.Aliquotout400µlportionsofthecolumneluateto1.5mlEppendorftubes.

23.Add1/10volumeof3Msodiumacetate(pH5.2).

24.Add1mlofethanoltoeachtube.

25.Letstandfor15minutes.

26.Centrifugeat12000xgat4Cfor15minutes.

27.Washpellet2timesin75%EtOHthenstoreat-80°C

RNACleanup

28.ResuspendRNAatapproximately1mg/mlinDEPCH2O.

29.Concentratetogreaterthan7mg/mlbycentrifugationonaMicroCon100filterunit,centrifugeat500xg,checkingasnecessarytodeterminetherateofconcentration.

Thisstepremovesmanyresidual,smalltomediumsized,moleculesthatinhibitthereversetranscriptionreactioninthepresenceoffluorescentlyderivatizednucleotides.

30.DeterminetheconcentrationofRNAintheconcentratedsamplebyspectrophotometry.Storeat-80°C.

SampleLABELINGbyReverseTranscription

1.IfusingananchoredoligodTprimer,annealtheprimertotheRNAinthefollowing17µlreaction(usea0.2mlthinwallPCRtubesothatincubationscanbecarriedoutinaPCRcycler):

IfusinganoligodT(12-18)primer,annealtheprimertotheRNAinthefollowing17µlreaction:

TheincorporationrateforCy5-dUTPislessthanthatofCy3-dUTP,somoreRNAislabeledtoachievemoreequivalentsignalfromeachspecies.

2.Heatto65°Cfor10minutesandcoolonicefor2minutes.

3.Add23µlofreactionmixturecontainingeitherCy5-dUTPorCy3-dUTPnucleotides,mixwellbypipettinganduseabriefcentrifugespintoconcentrateinthebottomofthetube:

Superscriptpolymeraseisverysensitivetodenaturationatair/liquidinterfaces,sobeverycarefultosuppressfoaminginallhandlingofthisreaction.

4.Incubateat42°Cfor30min.thenadd2µlSuperscriptII.Makesuretheenzymeiswellmixedinthereactionvolumeandincubateat42OCfor30-60min.

5.Add5µlof0.5MEDTA.

BesureyouhavestoppedyourreactionwithEDTAbeforeaddingNaOH,sincenucleicacidsprecipitateinalkalinemagnesiumsolutions.

6.Add10µl1NNaOH,incubateat65OCfor60minutestohydrolyzeresidualRNA.Cooltoroomtemperature.

Thepurityofthesodiumhydroxidesolutionusedinthisstepiscrucial.SlightcontaminationorlongstorageinaglassvesselcanproduceasolutionthatwilldegradetheCy5dyemolecule,turningthesolutionyellow.Someresearchersachievebetterresultsbyreducingthetimeofhydrolysisto30minutes.

7.Neutralizebyadding25µlof1MTris-HCl(pH7.5).

8.DesaltthelabeledcDNAbyaddingtheneutralizedreaction,400µlofTEpH7.5and20µgofhumanC0t-1DNAtoaMicroCon100cartridge.Pipettetomix,spinfor10minutesat500xg.

9.Washagainbyadding200µlTEpH7.5andconcentratingtoabout20-30µl(approximately8-10minat500xg).

Alternatively,asmallerporeMicroCon30canbeusedtospeedtheconcentrationstep.Inthiscase,centrifugethefirstwashforapproximately4.5minutesat16,000xgandthesecond(200µlwash)forabout2.5minutesat16,000xg.

10.Recoverbyinvertingtheconcentratoroveracleancollectiontubeandspinningfor3minat500xg.

Insomecases,thecy5labeledcDNAwillformagelatinousblueprecipitatethatisrecoveredintheconcentratedvolume.Thepresenceofthismaterialsignalsthepresenceofcontaminants.Themoreextremethecontamination,thegreaterthefractionofcDNAwhichwillbecapturedinthisgel.Evenifheatsolubilized,thismaterialtendstoproduceuniform,non-specificbindingtotheDNAtargets.

Whenconcentratingbycentrifugalfiltration,thetimesrequiredtoachievethedesiredfinalvolumearevariable.Overlylongspinscanremovenearlyallthewaterfromthesolutionbeingfiltered.Whenfluor-taggednucleicacidsareconcentratedontothefilterinthisfashion,theyareveryhardtoremove,soitisnecessarytoapproachthedesiredvolumebyconservativeapproximationsoftherequiredspintimes.Ifcontrolofvolumesprovesdifficult,thefinalconcentrationcanbeachievedbyevaporatingliquidinthespeed-vac.Vacuumevaporation,ifnottodryness,doesnotdegradetheperformanceofthelabeledcDNA.

11.Takea2-3µlaliquotoftheCy5labeledcDNAforanalysis,leaving18-28µlforhybridization.

12.Runthisprobeona2%agarosegel(6cmwidex8.5cmlong,2mmwideteeth)inTrisAcetateElectrophoresisBuffer(TAE).

Formaximalsensitivitywhenrunningsamplesonagelforfluoranalysis,useloadingbufferwithminimaldyeanddonotaddethidiumbromidetothegelorrunningbuffer.

13.ScanthegelonaMolecularDynamicsStormfluorescencescanner(setting:redfluorescence,200micronresolution,1000voltsonPMT)

Successfullabelingproducesadensesmearofprobefrom400bpto>1000bp,withlittlepile-upoflowmolecularweighttranscripts(asinFigure1,LaneA).Weaklabelingandsignificantlevelsoflowmolecularweightmaterialindicatesapoorlabeling(asinFigure1,LaneB).Afractionoftheobservedlowmolecularweightmaterialisunincorporatedfluornucleotide.

Hybridization

5.1HybridizefluorescentcDNAtoslide

1.Determinethevolumeofhybridizationsolutionrequired.Theruleofthumbistouse0.033µlforeachmm2ofslidesurfaceareacoveredbythecoverslipusedtocoverthearray.Anarraycoveredbya24mmby50mmcoverslipwillrequire40µlofhybridizationsolution.Thevolumeofthehybridizationsolutioniscritical.Whentoolittlesolutionisused,itisdifficulttoseatthecoverslipwithoutintroducingairbubblesoversomeportionofthearrayedESTs,andthecoverslipwillnotsitatauniformdistancefromtheslide.Ifthecoverslipisbowedtowardtheslideinthecenter,therewillbelesslabeledcDNAinthatareaandhybridizationwillbenon-uniform.Whentoomuchvolumeisapplied,thecoverslipwillmoveeasilyduringhandling,leadingtomisplacementrelativetothearrayedESTs,andnon-hybridizationinsomeareasofthearray.

2.Fora40µlhybridization,pooltheCy3andCy5labeledcDNAsintoasingle0.2mlthinwallPCRtubeandadjustthevolumeto30µlbyeitheraddingDEPCH2O,orremovingwaterinaSpeedVac.Ifusingavacuumdevicetoremovewater,donotusehighheatorheatlampstoaccelerateevaporation.Thefluorescentdyescouldbedegraded.

3.Fora40µlhybridizationcombinethefollowingcomponents:

Arraysandsamplescanvarysomewhat,makingitnecessarytovarythecompositionofthehybridizationcocktail.IncaseswherethereisresidualhybridizationtocontrolrepeatDNAsamplesonthearray,moreC0t-1DNAcanbeused,asintheHighSampleBlockingformulation.Whenthereisdiffusebackgroundorageneralhazeonallofthearrayelements,moreofthenon-specificblockercomponentscanbeused,asintheHighArrayBlockingformulation.

4.Mixthecomponentswellbypipetting,heatat98°Cfor2minutesinaPCRcycler,coolquicklyto25°Candadd0.6ulof10%SDS.

5.Centrifugefor5minat14,000xg.ThefluorlabeledcDNAshaveatendencytoformsmall,veryfluorescent,aggregateswhichresultinbright,punctatebackgroundonthearrayslide.Hardcentrifugationwillpellettheseaggregates,allowingyoutoavoidintroducingthemtothearray.

6.ApplythelabeledcDNAtoa24mmx50mmglasscoverslipandthentouchwiththeinvertedmicroarray.

Applyingthehybridizationmixtothearrayandcoverslippingitisanoperationwhichrequiressomedexteritytogetthepositioningofthecoverslipandtheexclusionofairbubblesjustright.Itishelpfultopracticethisoperationwithbufferandplainslidesbeforeattemptingactualsamples.Thehybridizationsolutionisaddedtothecoverslipfirst,sincesomeaggregatesoffluorremaininthesolutionandwillbindtothefirstsurfacetheytouch.

7.Placetheslideinamicroarrayhybridizationchamber,add5µlof3xSSCinthereservoir,ifthechamberprovidesone,oratthescribedendoftheslideandsealthechamber.Submergethechamberina65°Cwaterbathandallowtheslidetohybridizefor16-20hours.

Thereareawidevarietyofcommercialhybridizationchambers.Itisworthwhiletoprepareamockhybridizationwithablankslide,loaditinthechamberandincubateittotestforleaks,ordryingofthehybridizationfluid,eitherofwhichcauseseverefluorescentnoiseonthearray.

5.2WashoffunboundfluorescentcDNA

8.Removethehybridizationchamberfromthewaterbath,coolandcarefullydryoff.Unsealthechamberandremovetheslide.

Astheremaybenegativepressureinthechamberaftercooling,itisnecessarytoremovewaterfromaroundthesealssothatitisnotpulledintothechamberandontotheslidewhenthesealsareloosened.

9.Placetheslide,withthecoverslipstillaffixed,intoaCoplinjarfilledwith0.5XSSC/0.01%SDSwashbuffer.Allowthecoversliptofallfromtheslideandthenremovethecoverslipfromthejarwithaforceps.Allowtheslidetowashfor2-5minutes.

10.TransfertheslidetoafreshCoplinjarfilledwith0.06XSSC.Allowtheslidetowashfor2-5minutes.

Thesequenceofwashesmayneedtobeadjustedtoallowformoreaggressivenoiseremoval,dependingonthesourceofthesampleRNA.Usefulvariationsaretoaddafirstwashwhichis0.5XSSC/0.1%SDSortorepeatthenormalfirstwashtwice.

11.Transfertheslidetoasliderackandcentrifugeatlowrpm(700-1000)for3minutesinaclinicalcentrifugeequippedwithahorizontalrotorformicrotiterplates.

Iftheslideissimplyairdried,itfrequentlyacquiresafluorescenthaze.Centrifugingofftheliquidsresultsinalowerfluorescentbackground.Astherateofdryingcanbequiterapid,itissuggestedthattheslidebeplacedinthecentrifugeimmediatelyuponremovalfromtheCoplinjar.