The EpiQuik™m6ARNAMethylationQuantificationKit(Fluorometric) isacompletesetofoptimizedbuffersandreagentstofluorometricallyquantifyN6-methyladenosine(m6Aor6mA)inRNA.Itissuitableforadirectdetectionofm6A RNAmethylation statususingtotalRNAisolatedfromanyspeciessuchasmammals,plants,fungi,bacteria,andviruses.Thekithasthefollowingadvantagesandfeatures:

• Fluorometricassaywitheasy-to-followstepsforconvenienceandspeed.Theentireprocedurecanbecompletedwithin3hoursand45minutes.
• Highsensitivity,ofwhichthedetectionlimitcanbeaslowas5pgofm6A.
• UniquebindingsolutionallowsthatRNA>70ntscanbetightlyboundtothewells,whichenablesquantificationofm6AfrombothmRNAandnc-RNAsuchastRNA,rRNAandsnRNA.
• Optimizedantibodyandenhancersolutionsallowhighspecificitytom6A,withnocross-reactivitytounmethylatedadenosinewithintheindicatedconcentrationrangeofthesampleRNA.
• Universalpositiveandnegativecontrolsareincluded,whicharesuitableforquantifyingm6Afromanyspecies.
• Strip-wellmicroplateformatmakestheassayflexIBLeformanualorhighthroughputanalysis.
• Simple,reliable,andconsistentassayconditions.

BackgroundInformation 
N6-methyladenosine(m6A)isthemostcommonandabundantmodificationonRNAmoleculespresentineukaryotes.Them6AmodificationiscatalyzedbyamethyltransferasecomplexMETTL3andremovedbytherecentlydiscoveredm6ARNAdemethylasesFTOandALKBH5,whichcatalyzem6Ademethylationinanα-ketoglutarate(α-KG)-andFe2+-dependentmanner.ItwasshownthatMETTL3,FTOandALKBH5playimportantrolesinmanyBIOLOGicalprocesses,rangingfromdevelopmentandmetabolismtofertility.m6Aaccountsformorethan80%ofallRNAbasemethylationsandexistsinvariousspecies.m6AismainlydistributedinmRNAandalsooccursinnon-codingRNAsuchastRNA,rRNAandsnRNA.Therelativeabundanceofm6AinmRNAtranscriptshasbeenshowntoaffectRNAmetabolismprocessessuchassplicing,nuclearexport,translationABIlityandstabilityandRNAtranscription.Abnormalm6Amethylationlevelsinducedbydefectsinm6ARNAmethylaseanddemethylasecouldleadtodysfunctionofRNAandcausedisease.Forexample,abnormallylowlevelsofm6AintargetmRNAsduetoincreasedFTOactivityinpatientswithFTOmutations,throughanas-yetundefinedpathway,contributestotheonsetofobesityandrelateddiseases.Thedynamicandreversiblechemicalm6AmodificationonRNAmayalsoserveasanovelepigeneticMarkerofprofoundbiologicalsignificance.Therefore,moreusefulinformationforbetterunderstandingofm6ARNAmethylationlevelsanddistributiononRNAtranscriptscouldbenefitdiagnosticsandtherapeuticsofdisease.

Principle&Procedure
Thiskitcontainsallreagentsnecessaryforthequantificationofm6AinRNA.Inthisassay,totalRNAisboundtostripwellsusingRNAhighbindingsolution.m6Aisdetectedusingcaptureanddetectionantibodies.ThedetectedsignalisenhancedandthenquantifiedfluorometricallybyreADIngtheRFU(relativefluorescenceunits)withafluorescencespectrophotometer.Theamountofm6Aisproportionaltothefluorescenceintensitymeasured. 

StartingMaterials
Startingmaterialscanincludevarioustissueorcellsamplessuchascellsfromflaskormicroplateculturedcells,freshandfrozentissues,paraffin-embeddedtissues,blood,bodyfluidsamples,etc.

WB(10XWashBuffer)
BS(BindingSolution)
NC(NegativeControl,100µg/ml)*
PC(PositiveControl, m6A2µg/ml)*
CA(CaptureAntibody,1000X)*
DA(DetectionAntibody,1000X)*
ES(EnhancerSolution)*
FD(FluoroDeveloper)*
FE(FluoroEnhancer)*
DB(DilutionBuffer)
8-WellAssayStrips(WithFrame)

*Spinthesolutiondowntothebottompriortouse.