Genome-wide DNA methylation level analysis by micellar electrokinetic chromatography and laser-induced fluorescence detection after treatment of cell lines with azacytidine and antifolates

Abstract

Methylation of DNA is a well-known epigenetic mechanism to control DNA transcription. The determination of the exact methylation level of DNA samples is of great interest due to its significant deregulation in tumor cells. Here the genome-wide DNA methylation is quantified precisely using micellar electrokinetic chromatography (MEKC) combined with laser-induced fluorescence (LIF) detection after enzymatic DNA hydrolysis and coupling of the resulting mononucleotides with BODIPY FL EDA: N-[3-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen-3-yl)propionyl]ethylenediamine hydrochloride). For the validation of the method, two oligonucleotides containing 10 copies of each DNA base were designed and synthesized. In one oligonucleotides one cytosine residue was replaced with 5-methylcytosine, allowing the exact adjustment of different methylation levels between 0% and 10% by mixing appropriate amounts of these well-defined oligonucleotides. High precision, in particular of the detection factors of the single mononucleotides, was achieved because the complete analytical process, including hydrolysis, BODIPY coupling, and analysis, was considered during the calibration process. Application of this method on calf thymus DNA resulted in a methylation level of 6.94%, which is in good agreement with the values obtained with other methods. Whereas treatment of HEK293 cells with azacytidine led to considerably reduced global methylation from approximately 5.0% to 1.4%, treatment of the cells with the antifolates methotrexate and pemetrexed led to a slightly increased methylation level.