Measurement of DNA methylation using stable isotope dilution and gas chromatography-mass spectrometry.
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%.
Romerio
Antonella San
AS
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.
Fiorillo
Giovanni
G
Terruzzi
Ileana
I
Senesi
Pamela
P
Testolin
Giulio
G
Battezzati
Alberto
A
D004247:DNA / Q000032:analysis* / Q000737:chemistry*; D019175:DNA Methylation; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D015394:Molecular Structure; D011865:Radioisotope Dilution Technique
D004247:DNA