Development and validation of a gas chromatography/mass spectrometry method for the assessment of genomic DNA methylation.
A method for the determination of DNA global methylation, taken as the ratio (%) of 5-methylcytosine (5mCyt) versus the sum of cytosine (Cyt) and 5mCyt, via gas chromatography/mass spectrometry (GC/MS), was developed and validated. DNA (2.5 microg) was hydrolyzed with aqueous formic acid 88%, spiked with cytosine-2,4-(13)C(2),(15)N(3) and 5-methyl-(2)H(3)-cytosine-6-(2)H(1) as internal standards, and derivatized with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide and 1% tert-butyldimethylchlorosilane, in the presence of acetonitrile and pyridine. GC/MS, operating in single ion monitoring mode, separated and specifically detected all nucleobases as tert-butyldimethylsilyl derivatives, without interferences, with the exception of guanosine. The method was linear throughout the range of clinical interest and had good sensitivity, with a limit of quantification of 3.2 pmol for Cyt and 0.056 pmol for 5mCyt, the latter corresponding to a methylation level of 0.41%. Intra- and inter-day precision and accuracy were below 4.0% for both analytes and methylation. The matrix absolute effect, process efficiency and coefficient of variation ranged from 96.5 to 101.2%. The matrix relative effect was below 1%. The method was applied to the analysis of different human DNAs, including: nonmethylated DNA from PCR (methylation 0.00%), hypermethylated DNA prepared using M.SssI CpG methyltransferase (methylation 18.05%), DNA from peripheral blood leukocytes of healthy subjects (N = 6, median methylation 5.45%), DNA from bone marrow of leukemia patients (N = 5, 3.58%) and DNA from myeloma cell lines (N = 4, 2.74%).
Rossella
Federica
F
Department of Occupational and Environmental Medicine, University of Milano and Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Via S. Barnaba, 8, 20122 Milan, Italy.
Polledri
Elisa
E
Bollati
Valentina
V
Baccarelli
Andrea
A
Fustinoni
Silvia
S
D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry; D012680:Sensitivity and Specificity
D004247:DNA