Description:
MiRNAshavebeenwidelyknowntofunctionaseitheroncogenesortumorsuppressorsundercertainconditions.ThedysregulatedmiRNAshavebeenshowntoaffectthehallmarksofcancer,includingsustainingproliferativesignaling,eludinggrowthsuppressors,resistingcelldeath,andinducingangiogenesis.AnincreasingnumberofstudieshaveidentifiedmiRNAsaspotentialbioMarkersforhumancancerdiagnosis,prognosisandtherapeutictargets.SignosishasdevelopedCancermiRNAArrayincluding132cancer-relatedmiRNAsforcancerresearch.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 |
| miR-9-5p | miR-10a-5p | miR-15a-5p | miR-15b-5p | miR-16-5p | miR-17-5p | miR-18b-5p | miR-18b | 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-146b-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-199b-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 |
| miR-9-1-5p | miR-10b-5p | miR-17-3p | miR-22-3p | miR-23a-3p | miR-24-3p | miR-26a-5p | miR-26b-5p | miR-27a-3p | miR-27b-3p |
| miR-29a-3p | miR-29b-3p | miR-29c-3p | miR-30a-3p | miR-30a-5p | miR-30b-5p | miR-30c-5p | miR-92-3p | miR-92b-3p | miR-93-5p |
| miR-95-3p | miR-101-1-3p | miR-103-3p | miR-106a-5p | miR-106b-5p | miR-107 | miR-128a-3p | miR-128b-3p | miR-132-3p | miR-134-5p |
| miR-135b-5p | miR-136-5p | miR-137 | miR-140-5p | miR-141-3p | miR-142-3p | miR-149-5p | miR-150-5p | miR-151-3p | miR-153-3p |
| miR-154-5p | miR-181d-5p | miR-183-5p | miR-185-5p | miR-186-5p | miR-188-5p | miR-190-5p | miR-191-5p | miR-196a-5p | miR-196b-5p |
| miR-197-3p | miR-198 | miR-200b-3p | miR-202-5p | miR-203a | miR-205-5p | miR-210-3p | miR-214-3p | miR-215-5p | miR-218-5p |
| miR-219 | miR-221-3p | miR-222-3p | miR-296-5p | miR-372-3p | miR-373-5p | miR-488-5p | miR-100-5p | miR-127 | miR-142-5p |
| miR-31-5p | miR-181a-3p | RNU48 |
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:
HumanmiRNAarrayIIIanalysisofmiRNAexpressioninHeGP2,HEK293,andHeLacells.
