Bisulfite-Conversion and Pyrosequencing of ESR2 and H19

One-hundred nanograms of DNA was bisulfite-converted using the EpiTect Plus DNA Bisulfite Kit (Qiagen). PCR amplification of the ESR2 and H19 promoters was performed as previously described by different authors [15 (link),16 (link)] Bisulfite-converted DNA served as the template for the polymerase chain reaction (PCR) followed by pyrosequencing (PSQ). The PCR mix included PyroMark PCR Master Mix, 2×, and CoralLoad Concentrate, 10× (Qiagen), 0.2 µM of each primer, 1 µL of converted DNA, and nuclease-free water to a final volume of 25 µL. Primers used for DNA methylation analysis and PCR cycling conditions are shown in Table 1. The pyrosequencing reaction was run on a PyroMark Q96ID (Qiagen), and CpGs methylation analysis was conducted by the PyroMark CpG software (Qiagen). The methylation for each amplicon was calculated as the median of the methylation status of each analyzed CpG.