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 μg DNA analysis. In this range, healthy human DNA methylation percentage is within 5–6%.
Measurement of DNA methylation using stable isotope dilution and gas chromatography-mass spectrometry. / A. Sanromerio, G.Fiorillo, I. Teruzzi, P. Senesi, G. Testolin, A. Battezzati. - In: ANALYTICAL BIOCHEMISTRY. - ISSN 0003-2697. - 2005:336(2005 Jan), pp. 158-163.
Measurement of DNA methylation using stable isotope dilution and gas chromatography-mass spectrometry.
G.Fiorillo;I. Teruzzi;P. Senesi;G. Testolin;A. Battezzati
2005
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 μg DNA analysis. In this range, healthy human DNA methylation percentage is within 5–6%.
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