title: Global DNA methylation: comparison of enzymatic- and non-enzymatic-based methods.
abstract: BACKGROUND
  The most frequently used methods for measuring global DNA methylation are based on two different principles: the use of methylation-sensitive restriction endonucleases followed by analysis of the obtained fragments, or the hydrolysis of genomic DNA followed by specific detection and quantification of the 5-methylcytosine content. We aimed to compare two different methods for evaluation of global DNA methylation: the cytosine extension assay after enzymatic digestion of DNA (Cyt-Ext), and a recently described method using liquid chromatography-electrospray ionization-tandem mass spectrometry after DNA hydrolysis (LC-MS/MS).
  
  
  METHODS
  Both approaches were applied to evaluate global DNA methylation in leukocyte DNA from 96 healthy subjects. Calf thymus and pBR322 DNAs were used as hyper- and hypo-methylated references, respectively.
  
  
  RESULTS
  Using the Cyt-Ext method, the DNA from healthy individuals showed radiolabel incorporation of 11,312±1600 Dpm/μg DNA, while the LC-MS/MS method showed 4.55±0.1% methylation. Results are shown as mean±SD. The analysis of hypo- and hyper-methylated references showed that both methods are practical for discriminating different levels of methylation.
  
  
  CONCLUSIONS
  Cyt-Ext and LC-MS/MS are viable methods in evaluating global DNA methylation status. However, the LC-MS/MS assay allows absolute quantification and displays far superior intra-day precision. Therefore, we consider the later approach to be better for use in global DNA methylation studies.
authors.affiliation: Metabolism and Genetics Group (Met&Gen Group), Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Portugal. mesrocha@ff.ul.pt
authors.forename: Monica S
authors.initials: MS
authors.lastname: Rocha
authors.forename: Rita
authors.initials: R
authors.lastname: Castro
authors.forename: Isabel
authors.initials: I
authors.lastname: Rivera
authors.forename: Robert M
authors.initials: RM
authors.lastname: Kok
authors.forename: Yvo M
authors.initials: YM
authors.lastname: Smulders
authors.forename: Cornelis
authors.initials: C
authors.lastname: Jakobs
authors.forename: Isabel Tavares
authors.initials: IT
authors.lastname: de Almeida
authors.forename: Henk J
authors.initials: HJ
authors.lastname: Blom
mesh_terms: D044503:5-Methylcytosine / Q000032:analysis*; D002853:Chromatography, Liquid; D004247:DNA; D019175:DNA Methylation; D016678:Genome; D006801:Humans; D006868:Hydrolysis; D007962:Leukocytes; D008722:Methods; D015183:Restriction Mapping / Q000379:methods / Q000592:standards*; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D044503:5-Methylcytosine; D004247:DNA