The MethylFlash™ GlobalDNA Hydroxymethylation (5-hmC)ELISAEasyKit (Colorimetric) isacompletesetofoptimizedbuffersandreagentstocolorimetricallyquantifyglobalDNAhydroxymethylationstatusbyspecificallymeasuringlevelsof5-hydroxymethylcytosine(5-hmC)inasimplified,“one-step”ELISA-likeformat.AsafourthgenerationtechnologyofEpigentek"spopularglobalDNAmethylationtechnique,itisafurtherrefinementofthepredecessorMethylFlashkitbyimprovinguponspeed,simplicity,sensitivity,andreproducibility.

Thiskitisalsospecificallyoptimizedforpairedusewiththe MethylFlashGlobalDNAMethylation(5-mC)ELISAEasyKit(Colorimetric) forsimultaneouslyquantifyingbothmethylatedDNAandhydroxymethylatedDNA.

Thiskithasthefollowingadvantagesandfeatures:

• Fast -Reducedstepssothattheentireprocedureonlyneeds2hours*
• Robust -Improvedkitcompositionallowstheassaytohaveagreater“signalwindow”withreducedvariationbetweenreplicates
• Convenient -Inherentlylowbackgroundnoise,therebyeliminatingtheneedforDNAdenaturationandplateblockingsteps
• Sensitive -Detectionlimitcanbeaslowas0.01%hydroxymethylatedDNAfrom100ngofinputDNA
• Specific -Highspecificityto5-hmC,withnocross-reactivitytounmethylatedcytosineormethylatedcytosinewithintheindicatedconcentrationrangeofthesampleDNA
• Universal –PositiveandnegativecontrolsandallowdetectionofDNAhydroxymethylationinanyspeciesfromeithersingle-strandedordouble-strandedinputDNA
• Accurate -Optimizedpositivecontrolsthatcanbefractionalizedinpercentagescale,allowingtheassaytobemoreaccurateandhighlycomparablewithHPLC-MSanalysis
• FlexIBLe -Strip-wellmicroplateformatmakestheassayavailableformanualorhighthroughputanalysis

*Basedonasinglesampleassayinduplicate

Background
5-hydroxymethylcytosine(5-hmC),asasixthDNAbasewithfunctionsintranscriptionregulation,hasbeendetectedtobeabundantinhumanandmousebrainsandembryonicstem(ES)cells.Inmammals,itcanbegeneratedbytheoxidationof5-mC,areactionmediatedbytheten-eleventranslocation(TET)familyof5-mC-hydroxylases. 5-hmChasbeendemonstratedtobetissuespecific,rangingfromundetectablelevelsinculturedcelllinesto0.6%inhumanbraintissues,andcanbeashighas8%oftotalDNAinsomeotherspecies.TheBIOLOGicalsignificanceof5-hmCasanimportantepigeneticmodificationinphenotypeandgeneexpressionhasbeenrecentlyrecognized.Forexample,globaldecreasein5-hmCcontent(DNAhypomethylation)hasbeenexhibitedinnearlyallcancersandhasbeenproposedasamolecularMarkerandtherapeutictargetincanceraswell. 

Principle&Procedure
ThiskitcontainsallreagentsnecessaryforthequantificationofglobalDNAhydroxymethylation.Inthisassay,DNAisboundtostrip-wellsthatarespecificallytreatedtohaveahighDNAaffinity.ThehydroxymethylatedfractionofDNAisdetectedusinga5-hmCmAb-baseddetectioncomplexinaone-stepmannerandthenquantifiedcolorimetricallybyreADIngtheabsorbanceinamicroplatespectrophotometer.ThepercentageofhydroxymethylatedDNAisproportionaltotheODintensitymeasured.

StartingMaterials
InputDNAshouldberelativelypurewith260/280ratio>1.6andcanbedilutedwithwaterorTEbuffer.TheDNAamountcanrangefrom20ngto200ngperreaction.However,werecommendusing100ngofDNA,whichistheoptimizedinputamountforthebestresults. 

WB(10XWashBuffer)
BS(BindingSolution)
NC(NegativeControlwith0%5-hmC,50µg/ml)*
PC(PositiveControlcontains1%5-hmC,50µg/ml)*
hmAb(5-hmCAntibody,1000X)*
SI(SignalIndicator,1000X)*
ES(EnhancerSolution,1000X)*
DS(DeveloperSolution)
SS(StopSolution)
8-WellAssayStrips(WithFrame)
UserGuide

*Spinthesolutiondowntothebottompriortouse.

Note:TheNCisunhydroxymethylatedDNAcontaining0%of5-hydroxymethylcytosine.ThePC ishydroxymethylatedDNAcontaining1%of5-hydroxymethylcytosine.