Preparation of probes for in situ hybridization

• DNAoligonucleotidesarethepreferableprobesatpresent.Theyaresynthesizedchemically,whichallowsfortheincorporationofamino-modifiednucleotides(amino-allylT)atdefinedpositionsinthesequence.Thesefreeaminesarechemicallycoupledtofluorophoresaftersynthesis.ThemodifiedTsshouldbeabout10basesaparttopreventquenchingandhighbackground.TheGCcontentoftheoligoshouldbearound50%,itslengthcanbevariable,but40-50bases(5fluorochromespermoleculeofprobe)workswellinmostcases.ThesignalcanbefurtherincreasedbyusingseveraldifferentoligonucleotidesagainstthesameRNA.
• Aftersynthesis,theoligoshouldbepurifiedbyeithergelelectrophoresisorreversechromatographyonC-18Sep-Packcolumns(Waters).Oligosarethenconjugatedtoactivatedfluorophoresasdescribedbelow.

• RNAprobesaresynthesizedbyinvitrotranscriptionwithT3,T7orSp6RNApolymerases.

• Thefluorescentlabelcanbeincorporatedduringtranscription(followingmanufacturerindications),byusingnucleotidesthatarealreadycoupledtofluorophores.Unfortunately,thesenucleotidesareincorporatedwithalowfrequency,andtheresultingspecificactivityoftheseprobesisconsequentlysequencedependentandlow,i.e.lessthan1moleculeoffluorophoreper100basestranscribed.Furthermore,thesetranscriptionreactionscannotbephenolextracted,sincethefluorochromelabelledRNApartitionswiththephenolicphase.

• Alternatively,thefluorescentlabelcanbechemicallyconjugatedtotheRNAafterprobesynthesis,andthiscanresultinspecificactivitiesashighas1fluorophoreper10basestranscribed.Tothisend,RNAissynthesizedbystandardinvitrotranscriptionreactions,exceptthatUTPisreplacedbyanequalconcentrationofamino-allylUTP(Sigma),orbyamixtureofUTP/amino-allylUTP(usuallyata1/1ratio).Unlikefluorescentnucleotides,amino-allylnucleotidesareincorporatedalmostaswellasunmodifiedUTP.Transcriptionreactionsarethenphenolextracted,RNAisethanolprecipitated,resuspendedin1xSSC,andunincorporatednucleotidesareremovedbytworoundsofgelfiltration(1xSSCbufferedP30micro-spincolumn,BioRad).RNAisthenagainethanolprecipitatedandresuspendedinwater.

NB:ItisveryimportanttodesignRNAprobessuchthatnopolylinkersequencesarepresentintheresultingtranscript.ThepolylinkeroftencontainsGCrichstretchesthatinducecross-hybridizationwithribosomalRNA(WitkiewiczH,BolanderME,EdwardsDR,1993,Biotechniquesvol14,pp458-463).

• Thenucleicacidtobelabelledshouldberesuspendedin70µlof0.1MNaHCO3buffer,pH8.8.Differentamountsofmaterialwillresultindifferentspecificactivitiesoftheprobe.5-10µgofnucleicacidwillyieldahighspecificacitity(60-80%ofthefreeaminewillreact),while50µgwillyieldaloweractivity(5-20%ofthefreeaminewillbeconjugated).Labellingisinitiatedbyadding30µlofDMSOcontainingtheactivatedfluorophore.Inprinciple,anyaminereactivecompoundcouldbeused.Inpractice,CY3(1vialofmonoreactivelabellingkitperreaction,Amersham),worksverywellforasaredfluorophore,andthesuccinimidylesterofOregongreen488(1mgofcompoundperreaction,MolecularProbes)asagreendye.Labelingisthenconductedfor24-48hinthedark,atroomtemperature,withoccasionalvortexing.

NB:Anytraceoffreeamine,suchasTrisbase,shouldberemovedfromthenucleicacid,asitwillalsoreactwiththefluorophore.

• Unreacteddyeisthenremovedbyeithertworoundsofethanolprecipitationandwashing(carriertRNAcanbeaddedifneeded),orbygelfiltrationthroughG50columns.Specificactivityoftheprobesiscalculatedbyabsorptionspectroscopy.

NB:Itisusuallydesirabletoincorporateasmanyfluorophorespermoleculeofprobeaspossible,butitshouldbenotedthanmorethan4-5moleculesofdyeper40ntprobeoftenresultsinahighbackgroundduringinsituhybridization.