The MethylFlash™UrineN6-methyladenosine(m6A)QuantificationKit(Colorimetric) isacompletesetofoptimizedbuffersandreagentstocolorimetricallyquantifym6A inurineusinganinhibitorycompetitiveimmunoassaymethod.Itissuitablefordetectingtotalurinarym6A levels,resultingfromwholebodyturnoverordegradationofDNA/RNAcontainingm6A,usingurinefromhumansandanimals. Theurinesamplescanbeinfreshorfrozenform.Thekithasthefollowingadvantagesandfeatures:

• Innovativecolorimetricassaywitheasy-to-followstepsforconvenienceandspeed.Theentireprocedurecanbefinishedwithin4hours.
• 96strip-wellmicroplateformatmakestheassayflexIBLe:manualorhighthroughputanalysis.
• Innovativekitcompositionenablesbackgroundsignalstobeextremelylow,whicheliminatestheneedforplateblockingandallowstheassaytobesimple,accurate,reliable,andconsistent.
• Thelevelofm6AmeasuredinhumanurinesamplesusingthiskitiscomparabletothatdetectedbyHPLCmethod. 
• Anovelassayprincipleallowshighsensitivitytobeachieved.Thedetectionlimitcanbeaslowas0.01ng/assaywell.
• Lowinputrangeofurineforeachassaywithavolumeof1to20µlandanoptimalvolumeof5µl.
• Optimizedantibodyandenhancersolutionsallowforhighspecificitytom6A,withoutcross-reactivitytounmethylatedadenosine. 
• Negativecontrolandpositivestandardareincluded,whicharesuitableforquantificationofm6Ainfreeformandm6AcontainedinDNA/RNAfragmentsfromdifferenturinesamples.

BackgroundInformation 
Nucleobasem6A,amodifiedformofadenosineconvertedbyadenosinemethyltransferasesiswidespreadindifferentcellularRNAsandalsofoundinDNA.TheBIOLOGicalimportanceofDNA/RNAm6A-methylationasamajorepigeneticmodificationinphenotypeandgeneexpressionhasbeenrecognizedwidely.m6AplayscrucialrolesinregulatingDNAreplication,DNAdamage,RNAsplicing,transposition,transcription,andcellulardefense.Inhumans,them6AmodificationisprobablycatalyzedbyamethyltransferasecomplexMETTL3/METTL14andremovedbytheα-ketoglutarate(α-KG)-andFe2+-dependentdioxygenasessuchasFTO,ALKBH5andTET-likeenzymes.ItwasshownthatMETTL3andα-KG/Fe2+-dependentdioxygenasesplayimportantrolesinmanybiologicalprocesses,rangingfromdevelopmentandmetabolismtofertility. Urinaryexcretionofm6AisanindicationofthewholebodyturnoverorthedegradationofDNA/RNA,especiallytRNA.Theurinarym6Alevelcanbechangedwithachangeofthebodies’turnoverofm6ADNA/RNAoralterationofcellularDNA/RNAm6Astatus.Anumberofstudieshaveindicatedthatm6AexcretedinurinehasthepotentialtoactasacancerbioMarker.Forexample,anelevatedlevelofurinarym6Awasobservedincolorectalcancerpatientswithactivediseasestates. 

Principle&Procedure
InthisELISA-likeinhibitorycompetitiveimmunoassay,urinesamplesandthem6Astandardarefirstincubatedwitham6Aantibodysolutionandthentransferredtostripwellscoatedwithm6Apolynucleotide.ThewelliswashedtoremoveanyunboundreagentsafterincubationandthenadetectionantibodyisaddedtogenerateasignalthatcanbemeasuredcolorimetricallybyreADIngtheabsorbanceinamicroplatespectrophotometer.Becausem6Aintheurinesampleinhibitsthebindingofm6Aantibodytom6Acoatedonthewell,higherconcentrationsofm6Aintheurinesampleleadtoareducedbindingoftheantibodytothem6Aonthewell.ThereforethesignalorODintensitymeasuredfromthewellwillbeinverselyproportionaltotheamountofm6Aintheurinesampleandtheamountofm6Aintheurinesamplecanbequantifiedbyacomparisonwithapredeterminedm6Astandard.

StartingMaterials
Thevolumeofurineforeachassaycanbebetween1and20µl.Foroptimalquantification,theinputurinevolumeshouldbe5µl.

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.