TheMethylFlash™Urine5-Methylcytosine(5-mC)QuantificationKit (Colorimetric)  isacompletesetofoptimizedbuffersandreagentstocolorimetricallyquantify5-mCinurineusinganinhibitorycompetitiveimmunoassaymethod.It issuitablefordetectingtotalurinary5-methylcytosinelevels,resultingfromwholebodyturnoverordegradationofmethylatedDNA/RNA,inurinefromhumansandanimals.Theurinesamplescanbeinfreshorfrozenform. Thekithasthefollowingadvantagesandfeatures:

• Innovativecolorimetricassaywitheasy-to-followstepsforconvenienceandspeed.Theentireprocedurecanbefinishedwithin4hours.
• 96strip-wellmicroplateformatmakestheassayflexIBLe:manualorhighthroughputanalysis.
• Innovativekitcompositionenablesbackgroundsignalstobeextremelylow,whicheliminatestheneedforplateblockingandallowstheassaytobesimple,accurate,reliable,andconsistent.
• Thelevelof5-mCmeasuredinhumanurinesamplesusingthiskitiscomparabletothatdetectedbyHPLCmethod. 
• Anovelassayprincipleallowshighsensitivitytobeachieved.Thedetectionlimitcanbeaslowas0.03ng/assaywellor1nMof5-mC.
• Lowinputrangeofurineforeachassaywithavolumeof0.5to5µlandanoptimalvolumeof1µl.
• Optimizedantibodyandenhancersolutionsallowforhighspecificityto5-mC,withoutcross-reactivitytounmethylatedcytosine. 
• Negativecontrolandpositivestandardareincluded,whicharesuitableforquantificationof5-mCinfreeformand5-mCcontainedinmethylatedDNA/RNAfragmentsfromdifferenturinesamples.

BackgroundInformation
Nucleobase5-methylcytosine(5-mC),amodifiedformofcytosineconvertedbycytosinemethyltransferases,iswidespreadbothinDNAanddifferentcellularRNAs.TheBIOLOGicalimportanceofDNA5-mCmethylationasamajorepigeneticmodificationinphenotypeandgeneexpressionhasbeenwidelyrecognized.RecentdatastronglysuggeststhatRNA5-mCmethylationisalsoinvolvedintheregulationofvariousbiologicalprocessesincludingtRNAstABIlity/recognitionandmRNAtranslation. 

Urinaryexcretionof5-mCincludingboth5-methyl-2-deoxycytidineand5-methylcytidineisanindicationofawholebodyturnoverordegradationofmethylatedDNAandRNA.Theurinary5-mClevelcanbealteredbyachangeofthebodies’turnoverofmethylatedDNA/RNAoralterationofcellularDNA/RNAmethylationstatus.Anumberofstudieshaveindicatedthat5-mCexcretedinurinehasthepotentialtoactasacancerbioMarker,withanincreasedlevelarisingfromdiseaseonsetandprogression.Forexample,anelevatedlevelofurinary5-mCwasobservedinlungcancer,breastcancer,andleukemiapatientswithactivediseasestates.Itwasalsoshownthaturinary5-mCexcretioniseffectedbyAlzheimer’sdiseaseorbyrADIationtreatment.Itissuggestedthaturinary5-mCmightbeapplicableasabiologicalmarkerfordetectingsometypesofcancerandmonitoringcancerprogressionaftertreatmentwithradiationordemethylationreagents. 

Chromatography-basedtechniquessuchasHPLCandTLCmassspectrometryarecommonlyusedfordetecting5-mCinurineandalthoughfairlyacurate,aretimeconsuming,lesssensitive,andhavelowthroughputwithhighcosts.Toaddressthisproblem,EpigentekofferstheMethylFlash™ Urine5-Methylcytosine(5-mC)QuantificationKit (Colorimetric) toquantify5-mCorbodyturnoverstatusofmethylatedDNA/RNAusingurinesamples.

Principle&Procedure
InthisELISA-likeinhibitorycompetitiveimmunoassayassay,urinesamplesandthe5-mCstandardarefirstincubatedwitha5-mCantibodysolutionandthentransferredtostripwellscoatedwithmethylatedDNA(5-mC).Thewelliswashedtoremoveanyunboundreagentsafterincubationandthenadetectionantibodyisaddedtogenerateasignalthatcanbemeasuredcolorimetricallybyreadingtheabsorbanceinamicroplatespectrophotometer.Because5-mCintheurinesampleinhibitsthebindingof5-mCantibodyto5-mCcoatedonthewell,higherconcentrationsof5-mCintheurinesampleleadtoareducedbindingoftheantibodytothe5-mConthewell.ThereforethesignalorODintensitymeasuredfromthewellwillbeinverselyproportionaltotheamountof5-mCintheurinesampleandtheamountof5-mCintheurinesamplecanbequantifiedbyacomparisonwithapredetermined5-mCstandard.

Fig.1.SchematicprocedurefortheMethylFlash™Urine5-MethylcytosineQuantificationKit(Colorimetric).

Fig.2. 5-mCstandardwasaddedintotheassaywellsatdifferentconcentrationsandthenmeasuredwiththeMethylFlash™Urine5-MethylcytosineQuantificationKit (Colorimetric).

Fig.3. 5-mClevelisquantifiedfromdifferentvolumesofhumanurineusingtheMethylFlash™Urine5-MethylcytosineQuantificationKit (Colorimetric).