Measurement of DNA methylation using stable isotope dilution and gas chromatography-mass spectrometry |
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| Authors: | Romerio Antonella San Fiorillo Giovanni Terruzzi Ileana Senesi Pamela Testolin Giulio Battezzati Alberto |
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| Institution: | Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche (DiSTAM), International Center for the Assessment of Nutritional Status, Università degli Studi di Milano, 20133 Milan, Italy. |
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| Abstract: | A simple, highly selective, and sensitive method has been developed to quantify methylation of DNA extracted from human peripheral blood mononuclear cells. Assay has been performed at nucleobases level. Cytosine and 5-methylcytosine DNA content has been detected by gas chromatography-mass spectrometry using 2-(13)C]cytosine and 2-(13)C]5-methylcytosine as internal standards. The methylation level has been calculated as 5-methylcytosine/total cytosine ratio. The working range selected on calibration curve, obtained by evaluation of standards and matrix-added standards measurements, is suitable for 5 microg DNA analysis. In this range, healthy human DNA methylation percentage is within 5-6%. |
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| Keywords: | Cytosine 5-Methylcytosine Stable isotopes DNA methylation Gas chromatography-mass spectrometry |
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