2.5. DNA Isolation and Bisulfite Conversion
BLOOD
Blood samples taken at the beginning of the study were centrifuged at 4 °C for 15 min to obtain plasma and isolate the buffy coat fraction. DNA extraction was performed with the “MasterPure” DNA purification kit for blood version II (Epicentre Biotechnologies, Madison, WI, USA), and it was quantified with a spectrophotometer (Nanodrop, Thermo Scientific, Wilmington, DE, USA) and stored at −80 °C. In a second step, 500 ng of DNA was treated with sodium bisulfite using the EZ-96 DNA methylation kit (Zymo Research Corporation, Irvine, CA, USA), to convert unmethylated cytosine residues to uracil, while methylated cytokines remained unchanged.