Description:
NewlydiscoveredmicroRNAs(miRNAs)areimportanttotheregulationofgeneexpression,andupto30%ofmammaliangenesmayberegulatedbymiRNAs.Sofar,morethan400miRNAshavebeenidentifiedinthehumangenomeandmanyofthemareonlydifferentinoneorafewnucleotides.TheexpressionofmaturemiRNAsistissue-specificandtheabundanceofmiRNAsvariesoverseveralordersofmagnitude.Moreimportantly,mis-regulationofmiRNAexpressionmaycontributetohumancancers.SystematicprofilingofmiRNAexpressionplaysthekeyroleintheresearchofanumberofcancers.ApplicableGrid:
ListofApplicablemiRNAs
| let-7a-5p | let-7b-5p | let-7c-5p | let-7d-5p | let-7e-5p | let-7f-5p | let-7g-5p | let-7i-5p | miR-1 | miR-7-5p |
| miR-9-5p | miR-10a-5p | miR-15a-5p | miR-15b-5p | miR-16-5p | miR-17-5p | miR-18a-5p | miR-18b-5p | miR-19a-3p | miR-19b-3p |
| miR-20a-5p | miR-21-5p | miR-25-3p | miR-28-5p | miR-34a-5p | miR-99a-5p | miR-122a-5p | miR-124a-3p | miR-125a-5p | miR-125b-5p |
| miR-126-3p | miR-131 | miR-133a-3p | miR-133b | miR-143-3p | miR-145-5p | miR-146a-5p | miR-146a-5p | miR-148a-3p | miR-155-5p |
| miR-181a-5p | miR-181b-5p | miR-181c-5p | miR-182-5p | miR-192-5p | miR-194-5p | miR-195-5p | miR-199a-5p | miR-199a-5p | miR-199a*-3p |
| miR-200a-3p | miR-200c-3p | miR-204-5p | miR-206 | miR-216-5p | miR-223-3p | miR-224-5p | miR-342-3p | mir-376c-3p | miR-375 |
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:
miRNAanalysisofmiRNAexpressioninhumanbrainandliver.
5µgRNAwasincubatedandannealedwithDNAoligomix.DNA-RNAhybridswereselectedandligated.TheligatedmoleculeswereamplifiedandlabeledwithT7RNApolymeraseinthepresenceofbiotin-UTP.ThelabeledRNAswerehybridizedwithmiRNAarray, anddetectedwithchemiluminescenceimagingsystem.
