Quantification of DNA Methylation

Extraction of DNA was conducted using the conventional phenol/chloroform/isoamyl alcohol method. Hydrolysis of DNA was performed as previously described.19 (link) In brief DNA was hydrolyzed to nucleosides using nuclease P1, phosphodiesterase I and alkaline phosphatase (all from Sigma, St. Louis, MO, USA). Thereafter, internal standards were added to samples. The isotope-labeled internal standard for deoxycytidine was [¹⁵N₃]-2′-deoxycytidine, while that of 5-methyl-deoxycytidine and 5-hydroxymethyl-deoxycytidine were (methyl-d₃, ring-6-d₁)-5′-methyl-2′-deoxycytidine (both from Cambridge Isotopes Laboratories, Inc., Andover, MA, USA). The DNA hydrolysates were separated by an Agilent 1100 high-performance liquid chromatograph (Agilent Technologies, Palo Alto, CA, USA) and masses were detected through a 3200 Q Trap MS-MS system (Applied Biosystem, Concord, ON, Canada). Using the known masses of isotope-labeled internal standards added to each sample and the area of each peak the absolute amount of unmodified cytosine, 5′-methylcytosine and 5′-hydroxymethylcytosine per 1 μg of DNA can be calculated. Thereafter each amount was expressed as a percentage of total cytosine as we previously described.20 (link)