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.

Signosis基因功能分析，靶标发现和确认，分析开发以及化合物筛选通常需要基于细胞的分析。经过工程改造以通过转基因整合到宿主基因组中表达目的基因的稳定细胞系提供了进行此类分析的有效方法。然而，由于建立这种细胞系的过程繁琐，开发过程耗时，劳动，试剂和材料的昂贵以及单个克隆的转染后验证，是否值得仍然值得商de在单个实验室中开发这种稳定细胞系的方法。  为了促进研究，Signosis现在提供了一系列基因表达或荧光素酶报告基因就绪的稳定细胞系。 萤光素酶报道基因稳定细胞系EGFR稳定表达细胞系HER2（ERBB2）稳定表达的细胞系pLenti-MSCV-EGFR-GFP载体pCMV-EGFR载体pCMV-EGFR-IRES-GFP载体pMSCV-EGFR-GFP载体EGFR突变荧光素酶表达载体GAL4-UAS-luc报告基因稳定细胞系NR LBD驱动的GAL4 Reporter稳定细胞系亲代细胞系