Development and validation of a gas chromatography/ mass spectrometry method for the assessment of genomic DNA methylation

Federica Rossella, Elisa Polledri, Valentina Bollati, Andrea Baccarelli, Silvia Fustinoni

Research output: Contribution to journalArticle

Abstract

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 μg) was hydrolyzed with aqueous formic acid 88%, spiked with cytosine-2,4-13C2,15N3 and 5-methyl-2H3-cytosine-6-2H1 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 methyltransf erase (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%).

Original languageEnglish
Pages (from-to)2637-2646
Number of pages10
JournalRapid Communications in Mass Spectrometry
Volume23
Issue number17
DOIs
Publication statusPublished - Sep 15 2009

ASJC Scopus subject areas

  • Spectroscopy
  • Analytical Chemistry
  • Organic Chemistry

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