Description:
NumerousmiRNAshavebeenreportedtohaveaninfluenceontheregulationofapoptoticcellsignalingsuchastheoverexpressionofmiR-145,miR-216,miR-182,andmiR-96miRNAtodecreasecaspase-3activation.SignosishasdevelopedamiRNAarrayfortargeting52miRNAsthathavebeenshowninliteraturetobeinvolvedinapoptosis.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-10a-5p | miR-15a-5p | miR-15b-5p | miR-16-5p | miR-21-5p | miR-25-3p | miR-28-5p | miR-133b | miR-145-5p | miR-148 |
| miR-155-5p | miR-182-5p | miR-204-5p | miR-216-5p | miR-224-5p | mir-376c-3p | miR-24-3p | miR-101 | miR-142 | miR-150-5p |
| miR-151-3p | miR-153-3p | miR-188-5p | miR-196a-5p | miR-197-3p | miR-202-5p | miR-210-3p | miR-214-3p | miR-218-5p | miR-296-5p |
| miR-144 | miR-152 | miR-184 | miR-337 | miR-338 | miR-345 | miR-371 | miR-96 | miR-369-5p | miR-369-3p |
| miR-193a | miR-193b | RUN48 |
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:
Figure1.ApoptosismiRNAanalysis.
5µgoftotalRNAsfromU973,ap53negativecellline,andA431,ap53positivecellline,wereassayedwithhumanapoptosis miRNAarray anddetectedwithachemiluminescenceimagingsystem.TheinternalcontrolRNU48wasindicatedintheimage.
