The MethylFlash™ 5-mCRNAMethylationELISAEasyKit(Fluorometric) isacompletesetofoptimizedbuffersandreagentstofluorometricallyquantifyglobal 5-methylcytosine(5-mC) RNAmethylationlevels usingtotalRNAisolatedfromanyspeciesincludingmammals,plants,fungi,bacteria,andvirusesinavarietyofformsincluding,butnotlimitedto,culturedcells,freshandfrozentissues,plasma/serumsamplesandbodyfluidsamples,etc.Thiskit,basedonourpopularMethylFlash™quantificationtechnology,hasthefollowingadvantages:

• Fast -Theentireprocedureonlyneeds2hoursand40minutes
• Robust -Thekitcompositionallowstheassaytohavealarge“signalwindow”withlessvariationbetweenreplicates
• Convenient -Inherentlylowbackgroundnoise,therebyeliminatingtheneedforplateblockingsteps
• Sensitive -Detectionlimitcanbeaslowas0.02%of5-mCRNAfrom200ngofinputRNA
• Specific -Highspecificityto5-mC,withnocross-reactivitytounmethylatedcytosineorhydroxymethylatedcytosinewithintheindicatedconcentrationrangeofthesampleRNA
• Universal –Positiveandnegativecontrolsallowdetectionof5-mCRNAmethylationinanyspecies
• Accurate -Optimizedpositivecontrolsthatcanbefractionalizedinpercentagescale,allowingtheassaytobemoreaccurateandhighlycomparablewithHPLC-MSanalysis
• FlexIBLe -Strip-wellmicroplateformatmakestheassayavailableformanualorhighthroughputanalysis

BackgroundInformation
5-methylcytosine(5-mC)inDNAoccursbythecovalentadditionofamethylgroupatthe5-carbonofthecytosineringbyDNAmethyltransferases.Thisprocesshasbeenwellstudiedandisgenerallyassociatedwithrepressionofgeneexpression.Itwasalsoobservedthatinhumans,5-mCoccursinvariousRNAmoleculesincludingtRNAs,rRNAs,mRNAsandnon-codingRNAs(ncRNAs).5-mCseemstobeenrichedinsomeclassesofncRNA,butrelativelydepletedinmRNAs.Levelsof5-mCarevariableinanimalgenomes,rangingfromundetectableamountsinsomeinsectstoabout0.1-0.45%oftotalRNAinhumancells.Themajority(83%)of5-mCsiteswerefoundinmRNAs.Withinthesetranscripts5-mCappearstobedepletedwithinproteincodingsequencesbutenrichedin5’and3’UTRs.Twodifferentmethyltransferases,NSUN2andDnmt2areknowntocatalyze5-mCmodificationineukaryoticRNA.TherehasbeenstrongevidencethatRNAcytosinemethylationaffectstheregulationofvariousBIOLOGicalprocessessuchasRNAstABIlityandmRNAtranslation.FurThermore,lossof5-mCinvaultRNAscausesaberrantprocessingintoArgonaute-associatedsmallRNAfragmentsthatcanfunctionasmicroRNAs.Thus,impairedprocessingofvaultncRNAmaycontributetotheetiologyofhumandisordersrelatedtoNSun2-defciency. 

Principle&Procedure
Thiskitcontainsallreagentsnecessaryforthequantificationofglobal5-mCRNAmethylation.Intheassay,RNAisboundtostrip-wellsthatarespecificallytreatedtohaveahighnucleicacidaffinity.5-mCinRNAisdetectedusingcaptureanddetectionantibodiesandthenquantifiedfluorometricallybyreADIngthefluorescenceinamicroplatespectrophotometer.Thepercentageof5-mCRNAisproportionaltothefluorescenceintensitymeasured.

StartingMaterials
InputRNAshouldbehighlypurewith260/280ratio>2.0andrelativelyfreeofDNA.DNaseIcanbeusedtoremoveDNA.RNAshouldbeelutedinRNase-freewater.TheRNAamountcanrangefrom50ngto300ngperreaction.However,werecommendusing200ngofRNA,whichistheoptimizedinputamountforthebestresults.

WB(10XWashBuffer)
BS(BindingSolution)
NC(NegativeControlcontaining0%5-mC,50µg/ml)*
PC(PositiveControlcontaining2%5-mC,50µg/ml)*
mcAb(5-mCAntibody,1000X)*
SI(SignalIndicator,1000X)*
ES(EnhancerSolution,1000X)*
FD(FluoroDeveloper)
FE(FluoroEnhancer)
DB(Dilutionbuffer)
8-WellAssayStrips(WithFrame)
UserGuide

* Spinthesolutiondowntothebottompriortouse.