Global DNA methylation: Comparison of enzymatic- and non-enzymatic-based methods

Monica S. Rocha, Maria Rita Castro, Isabel Rivera, Robert M. Kok, Yvo M. Smulders, Cornelis Jakobs, Isabel Tavares De Almeida, Henk J. Blom

Research output: Contribution to journalArticle

7 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1793-1798
Number of pages6
JournalClinical Chemistry and Laboratory Medicine
Volume48
Issue number12
DOIs
Publication statusPublished - Dec 1 2010

    Fingerprint

All Science Journal Classification (ASJC) codes

  • Clinical Biochemistry
  • Biochemistry, medical

Cite this