TheEpiQuik™m6ARNAMethylationQuantificationKit(Colorimetric)isacompletesetofoptimizedbuffersandreagentstocolorimetricallyquantifyN6-methyladenosine(m6Aor6mA)inRNA.Itissuitableforadirectdetectionofm6ARNAmethylationstatususingtotalRNAisolatedfromanyspeciessuchasmammals,plants,fungi,bacteria,andviruses.Thekithasthefollowingadvantagesandfeatures: - ColorimetricELISA-likeassayusinganm6Aantibodywitheasy-to-followstepsforconvenienceandspeed.Theentireprocedurecanbecompletedwithin3hoursand45minutes.
- Highsensitivity,ofwhichthedetectionlimitcanbeaslowas10pgofm6A.
- UniquebindingsolutionallowsRNA>70ntstobetightlyboundtothewells,whichenablesquantificationofm6AfrombothmRNAandncRNAsuchastRNA,rRNA,andsnRNA.
- Optimizedantibodyandenhancersolutionsallowhighspecificitytom6A,withnocross-reactivitytounmethylatedadenosinewithintheindicatedconcentrationrangeofthesampleRNA.
- Universalpositiveandnegativecontrolsareincluded,whicharesuitableforquantifyingm6Afromanyspecies.
- Strip-wellmicroplateformatmakestheassayflexibleformanualorhighthroughputanalysis.
- Simple,reliable,andconsistentassayconditions.
BackgroundInformation
N6-methyladenosine(m6A)isthemostcommonandabundantmodificationinRNAmoleculespresentineukaryotes.Them6AmodificationiscatalyzedbyamethyltransferasecomplexMETTL3andremovedbytherecentlydiscoveredm6ARNAdemethylasesFTOandALKBH5,whichcatalyzem6Ademethylationinanα-ketoglutarate(α-KG)-andFe2+-dependentmanner.ItwasshownthatMETTL3,FTO,andALKBH5playimportantrolesinmanybiologicalprocesses,rangingfromdevelopmentandmetabolismtofertility.m6Aaccountsformorethan80%ofallRNAbasemethylationsandexistsinvariousspecies.m6AismainlydistributedinmRNAandalsooccursinnon-codingRNAsuchastRNA,rRNA,andsnRNA.Therelativeabundanceofm6AinmRNAtranscriptshasbeenshowntoaffectRNAmetabolismprocessessuchassplicing,nuclearexport,translationabilityandstability,andRNAtranscription.Abnormalm6Amethylationlevelsinducedbydefectsinm6ARNAmethylaseanddemethylasecouldleadtodysfunctionofRNAanddiseases.Forexample,abnormallylowlevelsofm6AintargetmRNAsduetoincreasedFTOactivityinpatientswithFTOmutations,throughanas-yetundefinedpathway,contributestotheonsetofobesityandrelateddiseases.Thedynamicandreversiblechemicalm6AmodificationinRNAmayalsoserveasanovelepigeneticMarkerofprofoundbiologicalsignificance.Therefore,moreusefulinformationforabetterunderstandingofm6ARNAmethylationlevelsanddistributiononRNAtranscriptscouldbenefitdiagnosticsandtherapeuticsofdisease. Principle&Procedure
Inthisassay,totalRNAisboundtostripwellsusingaRNAhighbindingsolution.N6-methyladenosine(m6A)isdetectedusingaspecificcaptureN6-methyladenosineantibodyanddetectionantibody.Thedetectedsignalisenhancedandthenquantifiedcolorimetricallybyreadingtheabsorbanceinamicroplatespectrophotometeratawavelengthof450nm.Theamountofm6AisproportionaltotheODintensitymeasured. BothnegativeandpositiveRNAcontrolsareprovidedinthiskit.Astandardcurvecanbeperformed(range:0.02to1ngofm6A)orasinglequantityofm6Acanbeusedasapositivecontrol.Becausem6Acontentcanvaryfromtissuetotissue,andfromnormalanddiseasedstates,orvaryundertreatedanduntreatedconditions,itisadvisedtorunreplicatesamplestoensurethatthesignalgeneratedisvalidated.Thiskitwillallowtheusertoquantifyanabsoluteamountofm6Aanddeterminetherelativem6ARNAmethylationstatesoftwodifferentRNAsamples. StartingMaterials
Startingmaterialscanincludevarioustissueorcellsamplessuchascellsfromflaskormicroplateculturedcells,freshandfrozentissues,paraffin-embeddedtissues,blood,bodyfluidsamples,etc. | 
 Fig.1. SchematicprocedureoftheEpiQuik™m6ARNAMethylationQuantificationKit(Colorimetric).
Fig.2. m6AstandardcontrolwasaddedintotheassaywellsatdifferentconcentrationsandthenmeasuredwiththeEpiQuik™m6ARNAMethylationQuantification Kit(Colorimetric). Fig.3. Quantificationofm6ARNAmethylationindifferentsamples. 200ngofRNAisolatedfromdifferenttissuesorcellswereaddedintotheassaywellsandthem6AcontainedinRNAwasmeasuredusingtheEpiQuik™m6ARNAMethylationQuantificationKit. |
