TheMethylFlash™MethylatedDNAQuantificationKit(Fluorometric)isacompletesetofoptimizedbuffersandreagentstofluorometricallyquantifyglobalDNAmethylationbyspecificallymeasuringlevelsof5-methylcytosine(5-mC)inamicroplate-basedformat. ComparedtoLUMAorLINE-1,Alu,andLTR-basedassays, MethylFlash™ technology directlyquantifiesactualglobalDNAmethylation. Thiskitisalsospecificallyoptimizedforpairedusewiththe MethylFlashHydroxymethylatedDNAQuantificationKit(Fluorometric) forsimultaneouslyquantifyingbothmethylatedDNAandhydroxymethylatedDNA. ProductFeatures
TheMethylFlash™MethylatedDNAQuantificationKit(Fluorometric)isafurtherrefinementofourpreviousSuperSense™globalDNAmethylationquantificationkitsby simplifyingtheworkflowandimprovingtheconsistencyofresults.It usesaninnovativemethodtoenablebackgroundsignalstobeextremelylow,eliminatingtheplatedryingandblockingsteps. Comparedtochromatography-basedmethodssuchasHPLCormassspectrometry,thisproductprovidesacost-effectivewaytoaccuratelymeasurelevelsof5-methylcytosine.Thekithasthefollowingadvantagesandfeatures: - Fluorometricassaywitheasy-to-followstepsforconvenienceandspeed.Theentireprocedurecanbefinishedwithin4hours.
- Innovativekitcompositionenablesbackgroundsignalstobeextremelylow,whicheliminatestheneedforplateblockingandallowstheassaytobesimple,accurate,reliable,andconsistent.
- Highsensitivity,ofwhichthedetectionlimitcanbeaslowas50pgofmethylatedDNAand acceptsaslowas20ngofinputgenomicDNA
- Optimizedantibodyandenhancersolutionsallowhighspecificityto5-mC,withnocross-reactivitytounmethylatedcytosineandnoornegligIBLecross-reactivitytohydroxymethylcytosinewithintheindicatedconcentrationrangeofthesampleDNA.
- Universalpositiveandnegativecontrolsareincluded,whicharesuitableforquantifyingmethylatedDNAfromanyspecies.
- Strip-wellmicroplateformatmakestheassayflexible:manualorhighthroughputanalysis.
Principle&Procedure
TheMethylFlash™MethylatedDNAQuantificationKit(Fluorometric)containsallreagentsnecessaryforthequantificationofglobalDNAmethylation.Inthisassay,DNAisboundtostripwellsthatarespecificallytreatedtohaveahighDNAaffinity.ThemethylatedfractionofDNAisdetectedusingcaptureanddetectionantibodiesandthenquantifiedfluorometricallybyreADIngtheabsorbanceinafluorescencemicroplatespectrophotometeratexcitation530andemission590nm.TheamountofmethylatedDNAisproportionaltotheRFU(relativefluorescenceunits)measured,whichcanbecalculatedwiththekit"sincludedformulasforrelativemethylationstatusoftwodifferentDNAsamplesorabsolutequantificationof5-methylcytosine(5-mC)usingastandardcurve. Easy,Fast,andFlexible
Theentirefluorometricassayhaseasy-to-followstepsforconvenienceandspeed,whichcanbecompletedin4hours.Thestrip-wellmicroplateformatallowsforaflexibleassayinmanualorhighthroughputanalysis.UniversalpositiveandnegativecontrolsareincludedwiththekitforquantifyingmethylatedDNAfromanyspeciessuchasmammals,plants,fungi,bacteria,andviruses. SafeandConvenient
Alltheneededreagents,includingnegativecontrolsandpositivecontrols,forquantificationofglobalDNAmethylationareconvenientlypackagedinthekit.ThedirectfluorometricquantificationofDNAsamplesreplacesobsoleteorinferiormethodsandeliminatestheneedforDNAdigestion/denaturation,radioactivity,extraction,orchromatography. HighlySensitiveandSpecific
ThenovelprocedureandproprietarykitcompositionsallowforaccuratequantificationofmethylatedDNAtobeachievedwithhighsensitivityandspecificity.ThedetectionlimitoftheinputDNAcanbeaslowas50pgofmethylatedDNA. ComparisonofAvailableELISA-basedGlobalDNAMethylationAssayMethods | | MethylFlash (Fluorometric) | CompetitorZ | CompetitorC |
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| AssayPrinciple | DirectELISA | DirectELISA | IndirectELISA | | Format | 96-wellplate | 96-wellplate | 96-wellplate | | Sensitivity | Excellentdetectionlimit:0.05ngof5-mCDNA | Gooddetectionlimit:>0.5ngof5-mCDNA | Verypoordetectionlimit:>1000ngof5-mCDNA | | S/NRatio | >20withverylowbackground | <4withhighbackground | N/A* | | ProceduralConvenience | NoneedforplateblockinganddenaturationofDNA | RequiresplateblockingandDNAdenaturation | Requiresplateblocking,DNAdigestion,andantibodycompetitiveincubationinanadditionalplate | | ProtocolTime | <4hr | <4hr | >8hr | | DNAType&Species | Universalforanyspecies,bothssDNAanddsDNA | HumanandmousessDNAonly | Variousspecies | | Specificity | Specificto5-mConly | Cross-reactionto5-hmC | N/A* | | QuantitationType | Bothrelativeandabsolutequantification | Relative | Relative | | StandardControl | Stable,quantitativeinabsoluteamountof5-mC,andcanbeuniversallyusedforanyspecies | Unstable,cannotbequantitativeinamount,andcanonlybeusedforhumanormouse | N/A* | | MinimumInputAmount | 20ng | 100ng | 1000ng | | AccuracyofDetection | High,stableandquantifiedstandardcontrol.ProvenclosecorrelationwithLC-MSanalysisbyusers | Unclear,unstableandun-quantifiedstandardcontrol | N/A* | | %5-mCcalculation | Simple | Complicated | NotProvided | | PatentedMethod | Yes | No | No | | Popularity | Veryhighpublishedcitationcount | Lowpublishedcitationcount | Lowpublishedcitationcount | | SupportExpertise | Since2006 | Since2013 | Since2011 |
*Insufficientinformationprovidedbydatasheetorproductfailedtoproducedatatomakeconclusivedetermination | 
 Fig.1.SchematicprocedurefortheMethylFlash™MethylatedDNAQuantificationKit(Fluorometric).
Fig.2. DemonstrationofhighsensitivityandspecificityofmethylatedDNAdetectionachievedbytheMethylFlash™kit.SyntheticunmethylatedDNA(contains50%ofcytosine)andmethylatedDNA(contains50%of5-methylcytosine)wereaddedintotheassaywellsatdifferentconcentrationsandthenmeasuredwiththeMethylFlash™MethylatedDNAQuantificationKit(Fluorometric).
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