TheMethylamp™RNABisulfiteConversionKitisacompletesetofoptimizedreagentsrequiredforfastbisulfiteconversiononaRNAsample.TheconvertedRNAobtainedwiththeMethylampRNABisulfite Conversion KitissuitableforvariousdownstreamRNAmethylationanalysesincludingmethylationspecificRT-PCR,MS-HRM,andbisulfiteRNA-sequencing(e.g.,pyrosequencinganddeep-sequencing).Thekithasthefollowingfeaturesandadvantages: - Fastandconvenientprotocolthatcanbefinishedin3hours.
- CompletelyconvertsunmethylatedRNAcytosineintouracil(>99.9%)withnoornegligIBLeinappropriate/errorconversionofmethylcytosinetothymine(<0.1%)whentheindicatedrangeofinputsampleRNAisused.
- PowerfulprotectionagainstRNAdegradation,withover90%ofRNAlossprevented.
- Includedcontrolprimersarespecificagainstbisulfite-convertedRNAandcanbeusedtotestwhetherthebisulfiteconversionhasbeenproperlyachieved.
- LowamountofinputRNAcanbeusedforbisulfiteconversionwithaslowas5ngperreaction.
- Simple,reliable,andconsistentreactionconditions.
BackgroundInformation
5-methylcytosine(5-mC)inDNAbeenwellstudiedandisgenerallyassociatedwithrepressionofgeneexpression,butithasalsobeenobservedthatinhumans,5-mCoccursinvariousRNAmoleculesincludingtRNAs,rRNAs,mRNAs,andnon-codingRNAs(ncRNAs).Atleast10,2755-mCcandidatesiteswerediscoveredinmRNAsandncRNAs,whichcovered10.6%ofthetotalcytosineresiduesinthetranscriptome.5-mCseemstobeenrichedinsomeclassesofncRNA,butrelativelydepletedinmRNAs.However,themajority(83%)oftheircandidatesiteswerefoundinmRNAs.Withinthesetranscripts,5-mCappearstobedepletedwithinproteincodingsequencesbutenrichedin5’and3’UTRs.Twodifferentmethyltransferases,NSUN2andDNMT2areknowntocatalyzethe5-mCmodificationineukaryoticRNAs.RecentdatastronglysuggestthatRNAcytosinemethylationaffectstheregulationofvariousBIOLOGicalprocessessuchasRNAstABIlityandmRNAtranslation.FurThermore,lossof5-mCinvaultRNAscausesaberrantprocessingintoArgonaute-associatedsmallRNAfragmentsthatcanfunctionasmicroRNAs.Thus,impairedprocessingofvaultncRNAmaycontributetotheetiologyofhumandisordersrelatedtoNSUN2-deficiencyhumandisorders. BisulfiteconversionofRNA,followedbyRT-PCRamplification,cloning,andsequencingyieldsreliableinformationaboutRNAcytosinemethylationstates.BytreatingRNAwithbisulfite,cytosineresiduesaredeaminatedtouracilwhileleaving5-methylcytosineintact: 
 Fig.2. BisulfiteconversionofRNA,followedbyRT-PCRamplification,cloning,andsequencing,yieldsreliableinformationaboutRNAcytosinemethylationstates.BytreatingRNAwithbisulfite,cytosineresiduesaredeaminatedtouracilwhileleaving5-methylcytosineintact.
Fig.3. RNAbisulfitesequencinganalysis.RNAisolated fromMCF-7cellsissubjecttobisulfitetreatment,CDNAsynthesisandsequencingafterPCRamplificationusing28SribosomalRNAprimersspecifictobisulfiteconvertedRNA.Unmethylatedcytosines(#4,#5,#14)areconvertedtouracilanddetectedasthyminewhilemethylatedcytosine(#8)remainsthesame. Principle&Procedure
TheMethylampRNABisulfiteConversionKitcontainsallreagentsrequiredforfastand efficientbisulfiteconversiononaRNAsample.AuniqueconversionmixsolutioncontainspowerfulRNAprotectionreagentstopreventchemicalandthermophilicdegradation,thusleADIngtoanacceleratedconversionofallcytosinestouracilwithnegligiblemethylcytosinedeamination.Thenon-toxicRNAcapturesolutionenablesRNAtotightlybindtothecolumnfilter,sothatconvertedRNAcleaningcanbecarriedoutonthecolumntoeffectivelyremoveresidualbisulfiteandsalts. StartingMaterials
RNAisolatedfromvarioustissueorcellsamplescanbeusedasstartingmaterial.TheamountofRNAforeachbisulfitereactioncanbe5ngto1µg.Foranoptimalreaction,theinputRNAamountshouldbe200ngto500ng.WhenusingtheMethylampRNABisulfiteConversionKitformethylation-specificRT-PCRwithverysmallamountsofinputRNA(<10ng),thenumberofPCRcyclesshouldbegreaterthan45.TheyieldofRNApurifiedafterbisulfiteconversiondependsontheamountofinputRNA,natureofRNA,andsourceofthestartingmaterial. | 
Fig.1. SchematicprocedureoftheMethylampRNABisulfiteConversionKittoobtainconvertedRNA.
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