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基因表达  是利用来自基因的信息指导功能性基因产物合成的过程。这些产品通常是蛋白质。基因表达分析对于理解基因及其蛋白产物的功能，以及对影响基本生物学过程的复杂调控网络的清晰了解至关重要。Signosis  提供了许多用于基因表达研究的测定产品，包括用于监控由TF上调或下调的基因表达的cDNA板阵列，以及用于分析基因表达的单细胞PCR试剂盒，可直接分析细胞无需RNA分离即可进行裂解。 SARS-CoV-2实时PCR检测试剂盒单细胞基因表达分析印迹分析RNA的cDNA板阵列细胞裂解液的cDNA板阵列