Werecommendusinganewerversionofthisproduct: MethylFlashGlobalDNAHydroxymethylation(5-hmC)ELISAEasyKit(Colorimetric) 

The MethylFlash™HydroxymethylatedDNAQuantificationKit(Colorimetric) isacompletesetofoptimizedbuffersandreagentstocolorimetricallyquantifyglobalDNAhydroxymethylationbyspecificallymeasuringlevelsof5-hydroxymethylcytosine(5-hmC)withoutcross-reactivitytomethylcytosineandunmethylatedcytosine,inanELISA-likemicroplate-basedformat.

Thiskitisalsospecificallyoptimizedforpairedusewiththe MethylFlashMethylatedDNAQuantificationKit(Colorimetric) forsimultaneouslyquantifyingbothmethylatedDNAandhydroxymethylatedDNA.

About5-Hydroxymethylcytosine
5-hmCisamodifiedformofcytosine, recentlydiscovered inanimaltissues.Thefunctionof5-hmCinepigeneticsmaybedifferentfromitsforerunner5-methylcytosine(5-mC)andcurrentlyremainsamystery.Itisbelieved,though,that5-hmCplaysanimportantroleinswitchinggenesonandoff. Thepresenceof5-hmCmakesitnecessarytonotonlyre-evaluateexistingDNAmethylationdata,butalsonecessarytodeterminetherelativedistributionandchangesof5-hmCinhumantissuesofhealthyanddiseasedstatuses.PriortoEpigentek’sMethylFlash™ technology,therewere nomethods thatcouldbeusedforpracticallyandroutinelyidentifying5-hmCanddiscriminatingthisbasefrom5-mC.

DistinguishingBetween5-hmCand5-mC
CurrentlyusedmethylatedDNAanalysismethodsincludingrestrictionenzymedigestionandbisulfiteorMeDIP-mediatedMS-PCRandsequencingarenotsuitablefor5-hmCdetectionas5-hmCand5-mCarevirtuallyindistinguishablebetweeneachotherwiththesemethods.Toaddressthisproblem,EpigentekofferstheMethylFlash™HydroxymethylatedDNAQuantificationKitwhichusesauniqueproceduretoquantifyglobalDNAhydroxymethylation. Thisproductprovidesacost-effectivewaytomeasurelevelsof5-hydroxymethylcytosineandtodistinguishbetween5hmC,5mC,andC.Thisallowsforresearcherstore-evaluatetheirDNAmethylationdataforDNAhydroxymethylationandtoefficientlylookforDNAhydroxymethylationinnewDNAsamples. Thekithasthefollowingadvantagesandfeatures:

• Colorimetricassaywitheasy-to-followstepsforconvenienceandspeed.Theentireprocedurecanbecompletedwithin3hoursand45minutes.
• Highsensitivity,ofwhichthedetectionlimitcanbeaslowas40pgofhydroxymethylatedDNA andacceptsaslowas50ngofinputgenomicDNA.
• Highspecificitywithnocross-reactivityto methylcytosineand unmethylatedcytosine.5-hydroxymethylcytosineisseparatelydetected. 
• Universalpositiveandnegativecontrolsareincluded,whicharesuitableforquantifyinghydroxymethylatedDNAfromanyspecies.
• Strip-wellmicroplateformatmakestheassayflexIBLeforeithermanualorhighthroughputanalysis.
• Simple,reliable,andconsistentassayconditions.

ComparisonofAvailableELISA-basedGlobalDNAMethylationAssayMethods 

HC1(10XWashBuffer)
HC2(BindingSolution)
HC3(NegativeControlI,20µg/ml)*
HC4(NegativeControlII,20µg/ml)*
HC5(PositiveControl,20µg/ml)*
HC6(CaptureAntibody,1000µg/ml*
HC7(DetectionAntibody,400µg/ml)*
HC8(EnhancerSolution)*
HC9(DeveloperSolution)
HC10(StopSolution)
8-WellAssayStrips(WithFrame)
UserGuide

*Spinthesolutiondowntothebottompriortouse.

Note:TheHC3NegativeControlIisanunmethylatedpolynucleotidecontaining20%ofcytosine.TheHC4NegativeControlIIisamethylatedpolynucleotidecontaining20%of5-methylcytosine.TheHC5PositiveControlisahydroxymethylatedpolynucleotidecontaining20%ofhydroxymethylcytosine.