DNA was extracted from fresh-frozen tissue samples following the standard phenol-chloroform purification protocol as described previously [10]. Four hundred ng of purified DNA were bisulfite-modified, using the EZ DNA Methylation™ Kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s instructions, and analyzed by methylation-specific PCR (MSP). MSP primers, overlapping with the location of the microarray probes of interest, were designed with Methyl Primer Express Software v1.0 (Applied Biosystems™, Thermo Fisher Scientific, Carlsbad, CA, USA) and ordered from Metabion (Martinsried, Germany) (Table S2). The reaction mix (25 μL) consisted of 1× Maxima Hot Start Taq PCR buffer, 2.5 mM MgCl2, 0.4 mM of each dNTP, 1.25 U Maxima Hot Start Taq DNA Polymerase (all from Thermo Scientific™, Thermo Fisher Scientific, Vilnius, Lithuania), 1 µM of each primer, and 10–20 ng of bisulfite-treated DNA. Reaction conditions were optimized before the study and included 35–38 cycles with primer annealing step 55–62 °C for 45 s (Table S2). Methylation-positive (MC), methylation-negative, and non-template controls (NTC) were included in each MSP assay.

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