Description:
Signosis"HumanmiRNAarrayIVallowsyoutoprofile100miRNAs.ApplicableGrid:
ListofApplicablemiRNAs
| miR-138 | miR-139 | miR-105 | miR-130a | miR-135a | miR-147 | miR-152 | miR-154-3p | miR-184 | miR-187 |
| miR-189 | miR-211 | miR-212 | miR-217 | miR-22 | miR-220 | miR-299-3p | miR-299-5p | miR-301 | miR-302a-3p |
| miR-302a-5p | miR-302b-3p | miR-302b-5p | miR-302c-3p | miR-302c-5p | miR-302d | miR-30d | miR-30e-3p | miR-30e-5p | miR-32 |
| miR-320 | miR-323 | miR-324-3p | miR-324-5p | miR-325 | miR-326 | miR-328 | miR-329 | miR-33 | miR-330 |
| miR-331 | miR-335 | miR-337 | miR-338 | miR-339 | miR-33b | miR-340 | miR-345 | miR-346 | miR-34b |
| miR-34c | miR-361 | miR-362 | miR-363-3p | miR-363-5p | miR-365 | miR-367 | miR-369-3p | miR-369-5p | miR-370 |
| miR-371 | miR-373-5p | miR-374 | miR-376a-3p | miR-376a-5p | miR-376b | miR-377 | miR-378 | miR-379 | miR-380-3p |
| miR-380-5p | miR-381 | miR-382 | miR-383 | miR-384 | miR-409-5p | miR-410 | miR-411 | miR-412 | miR-422a |
| miR-422b | miR-423 | miR-424 | miR-425 | miR-425-5p | miR-429 | miR-432-5p | miR-432-3p | miR-433 | miR-448 |
| miR-449 | miR-449b | miR-450 | miR-451 | miR-452-5p | miR-452-3p | miR-453 | miR-455 | miR-483 | U6 |
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:
