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Global DNA methylation: comparison of enzymatic- and non-enzymatic-based methods
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Monica S. Rocha
Published/Copyright:
October 28, 2010
Abstract
Background: The most frequently used methods for meas-uring 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.
Clin Chem Lab Med 2010;48:1793–8.
Received: 2009-12-7
Accepted: 2010-6-10
Published Online: 2010-10-28
Published in Print: 2010-12-01
©2010 by Walter de Gruyter Berlin New York
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