The MethylFlash™5-Formylcytosine(5-fC)DNAQuantificationKit(Colorimetric) isacompletesetofoptimizedbuffersandreagentstocolorimetricallyquantify5-formylcytosine(5-fC)inamicroplate-basedformat.ItissuitableforusewithDNAisolatedfromanyspecies(mammals,plants,fungi,bacteria,viruses,etc.)inavarietyofformsincluding,butnotlimitedto,culturedcells,freshandfrozentissues,paraffin-embeddedtissues,andbodyfluidsamples.Thekithasthefollowingadvantagesandfeatures:

• Fastandeasyprocedurecompletedwithinjust3hoursand45minutes.
• Highsensitivity,ofwhichthedetectionlimitcanbeaslowas1pgof5-fC.
• Highspecificitywithdetectionofonly5-fC.Nocross-reactivitytocytosine,5-mC,5-hmC,or5-caCwithintheindicatedconcentrationrangeofthesampleDNA.
• HighlyconvenientassaythatusesDNAisolatedfromcellsortissuesdirectly;noneedforDNAdigestionorhydrolysis.
• Universalpositiveandnegativecontrolsareincluded,whicharesuitableforquantifying5-fCfromanyspecies. 
• Strip-wellmicroplateformatmakestheassayflexIBLe:manualorhighthroughputanalysis.
• Simple,reliable,andconsistentassayconditions. 

BackgroundInformation
5-formylcytosine(5-fC)hasrecentlybeenfoundinmammaliantissueandcellsandhasbeenclassifiedasaseventhDNAbase.Itisformedbytheoxidationof5-hydroxymethylcytosinethroughTEThydroxylasesandhasbeendemonstratedtobeanintermediateoftheDNAdemethylationprocess.Thefunctionof5-fCingeneregulationisnotyetentirelyclear,butithasbeenshownthat5-fCexhibitsreplication-dependentdilutionduringmousepre-implantationdevelopmentandcouldbefunctionalinregulatingpre-implantationdevelopmentasawhole.Thedetectionof5-fCinvarioustissuesandcellsisimportantbecause5-fCcouldbeaMarkertoindicateactiveDNAdemethylation.5-fCcanalsobedirectlyexcisedbyThymineDNAglycosylase(TDG)toallowsubsequentbaseexcisionrepair(BER)processingwhichconvertsmodifiedcytosinebacktoitsunmodifiedstate.

Severalchromatography-basedtechniquessuchasHPLC-MSareusedforthedetectionof5-fCintissueandcells.However,thesemethodsaretimeconsuming,requirelargeamountsofDNAandhavelowthroughputwithhighcosts.Toaddresstheseproblems,EpigentekofferstheMethylFlash™5-Formylcytosine(5-fC)DNAQuantificationKit(Colorimetric),whichusesauniquemicroplate-basedproceduretodirectlyquantify5-fC.

Principle&Procedure
TheMethylFlash™5-Formylcytosine(5-fC)DNAQuantificationKit(Colorimetric)containsallthereagentsnecessaryforthequantificationof5-fC.Inthisassay,DNAisboundtostrip-wellsthatarespecificallytreatedtohaveahighDNAaffinity.5-fCisdetectedusingcaptureanddetectionantibodies.ThedetectedsignalisenhancedandthenquantifiedcolorimetricallybyreADIngtheabsorbanceinamicroplatespectrophotometeratawavelengthof450nm.Theamountof5-fCisproportionaltotheODintensitymeasured.

StartingMaterials&InputAmount
ThedetectionlimitoftheinputDNAcanbeaslowas1pgof5-fCwithadynamicrangefrom10pgto400pg(seeFig.2).Thereisnocross-reactivitytocytosine,5-mC,or5-hmCwithintheindicatedconcentrationrangeofthesampleDNA(seeFig.3). 

SafeandConvenient
Allthenecessaryreagents,includingnegativeandpositivecontrols,forthequantificationof5-fCareconvenientlypackagedinthekit.ThedirectcolorimetricquantificationofDNAsamplesina96-wellmicroplateformateliminatestheneedforDNAdigestion/denaturation,radioactivity,extraction,orchromatography.

Easy,Fast,andFlexible
Theentirecolorimetricassayhaseasy-to-followstepsforconvenienceandspeed,allowingittobecompletedinjust3hoursand45minutes.Thestrip-wellmicroplateformatallowsforaflexibleassayformanualorhighthroughputanalysis.Universalpositiveandnegativecontrolsareincludedwiththekitforthequantificationof5-fCfromanyspecies,suchasmammals,plants,fungi,bacteria,andviruses.

Responsive,Reliable,andPractical
Basedonitsworkingprincipleandthemicroplateformat,thekitcanbepracticallyandroutinelyusedforanyspeciesandforavarietyofformsincludingculturedcells,freshandfrozentissues,andparaffin-embeddedtissues.TodemonstratethecapABIlitiesofthekit,ithasbeensuccessfullyusedforquantifyingthecontentof5-fCinDNAfromhumanbraintissue, mousebraintissue, rectaltissue,andrectalcancer.Thepercentageof5-fCmeasuredbythekitisparallelandcomparabletothatdetectedbyLC-MS/MSmethods(seeFig.4).

WB(10XWashBuffer)
BS(BindingSolution)
NC(NegativeControl,10µg/ml)*
PC(PositiveControl,5-fC1µg/ml)*
CA(CaptureAntibody,1000X)*
DA(DetectionAntibody,2000X)*
ES(EnhancerSolution)*
DS(DeveloperSolution)
SS(StopSolution)
8-WellAssayStrips(WithFrame)
UserGuide

*Spinthesolutiondowntothebottompriortouse.