Description:
MiRNAsarefoundtobedifferentiallyexpressedinstemcells,suchasbrain-specificmiR-124aandmiR-9moleculesduringneurallineagedifferentiation.Inaddition,miRNAsarealsoreportedtoregulatecancerstemcells.SignosishasdevelopedamiRNAarrayspecificallytotarget69miRNAsthatareshowninliteraturetobeassociatedwithstemcelldifferentiationandmaintenance.Thearraycanbeusedforhuman,mouseandratsamples.Principle:
miRNAisdifferentfromlargemessengerRNAinthreeaspects;(1)miRNAsaresmallsizemoleculeswithquiteabigdifferenceinabundance,(2)maturemiRNAsco-existwiththeirprecursorpre-miRNAandpri-miRNA,whichonlydifferinlength,and(3)manymiRNAsareverycloselyrelatedinsequences,suchasisoforms,onlydifferingbyoneorafewnucleotides.Therefore,theconventionalmircoarraytechnologiescannotdirectlybeappliedtoanalyzingthesemolecules.AnumberofmiRNAmicroarrayproductsarecommerciallyavailable,buttheyareeithertediousinrequiringpre-isolationofmicroRNA,lackthediscriminativepowertodifferentiatebetweenisoforms,orarenotsensitiveenoughtomonitorlowabundantmiRNAs.
Inourarrayassay,eachmiRNAmoleculeistargetedbytwooligosthathybridizewiththetargetmiRNAtoformaRNA/DNAduplex.Whenthesequencesareperfectlymatched,theoligosarealignedwiththemiRNAandthejointcanbeligatedbyDNAligase(figure1).AsinglenucleotidedifferenceamongmiRNAswillblockeitherthehybridizationortheligation;Thus,miRNAisoformscanbedifferentiated.DuetothesmallsizeofmiRNA,thehybridmightnotbestable;Therefore,weintroducethestackingsequences.Byextendingthesetwooligosalongwiththeircomplementaryoligos,thestABIlityisincreased.Oncethepairofoligosisligated,theligatedmoleculesaresubjectedtolinearamplificationviaT7transcriptionintoRNAinthepresenceofbiotin-UTP,whichareusedasprobesforarrayhybridization.Todifferentiateeachisoform,weassigneduniquetagsequencestotheligationoligos,sothatsinglenucleotidedifferencesareconvertedintouniquetagsequences.Inthisway,eachisoformcanbeeasilydistinguishedbyarrayhybridization.
Theassayprocedureincludesthreesteps:(1)mixthemiRNAwithprovidedoligostoformmiRNA/oligohybrids;(2)selectthehybridsandremovefreeoligos,andligatemiRNA-directedpairingofoligostobecomeasingleDNA;and(3)amplifytheligatedDNAwithT7transcription.
Data:
Stemcell miRNAarray analysisofmiRNAexpressioninmouse (left)andmouseembroy(right). |
