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 Citations (Scopus)

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
StatePublished - Dec 1 2010

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DNA Methylation
Methylation
DNA
Hydrolysis
Assays
5-Methylcytosine
Electrospray ionization
Cytosine
DNA Restriction Enzymes
Liquid chromatography
Mass spectrometry
Electrospray Ionization Mass Spectrometry
Enzyme Assays
Tandem Mass Spectrometry
Liquid Chromatography
Digestion
Healthy Volunteers
Leukocytes

All Science Journal Classification (ASJC) codes

  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

Rocha, Monica S. ; Castro, Maria Rita ; Rivera, Isabel ; Kok, Robert M. ; Smulders, Yvo M. ; Jakobs, Cornelis ; De Almeida, Isabel Tavares ; Blom, Henk J. / Global DNA methylation : Comparison of enzymatic- and non-enzymatic-based methods. In: Clinical Chemistry and Laboratory Medicine. 2010 ; Vol. 48, No. 12. pp. 1793-1798.
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Rocha, MS, Castro, MR, Rivera, I, Kok, RM, Smulders, YM, Jakobs, C, De Almeida, IT & Blom, HJ 2010, 'Global DNA methylation: Comparison of enzymatic- and non-enzymatic-based methods', Clinical Chemistry and Laboratory Medicine, vol. 48, no. 12, pp. 1793-1798. https://doi.org/10.1515/CCLM.2010.346

Global DNA methylation : Comparison of enzymatic- and non-enzymatic-based methods. / Rocha, Monica S.; Castro, Maria Rita; Rivera, Isabel; Kok, Robert M.; Smulders, Yvo M.; Jakobs, Cornelis; De Almeida, Isabel Tavares; Blom, Henk J.

In: Clinical Chemistry and Laboratory Medicine, Vol. 48, No. 12, 01.12.2010, p. 1793-1798.

Research output: Contribution to journalArticle

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T2 - Comparison of enzymatic- and non-enzymatic-based methods

AU - Rocha, Monica S.

AU - Castro, Maria Rita

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AU - Kok, Robert M.

AU - Smulders, Yvo M.

AU - Jakobs, Cornelis

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AU - Blom, Henk J.

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