1 µg of EcoRI digested DNA was subjected to bisulfite treatment as previously described [105]. Loci were selected for further analysis from the locations of probes whose normalized intensities were significantly different between the groups (see Microarray Analysis). PCR amplifications were performed in two steps: 25 ng of bisulfite DNA were used for the outside PCR and 1 µl of this DNA for the nested PCR. The outside and nested PCR were performed using HotStarTaq DNA polymerase (Qiagen, Germantown, United States). For pyrosequencing analysis, 25 µl of the nested bisulfite-PCR products were processed according to the manufacturer’s standard protocol (Biotage, Charlotte, United States). Sequencing reactions were performed with a PyroMark Gold Q24 Reagent Kit (Qiagen (Biotage), Germantown, United States) according to the manufacturer’s instructions. The percentage methylation at each CpG site was calculated from the raw data by use of PyroMark Q24-CpG Software (Biotage, Charlotte, United States). The mean methylation of all CG sites analyzed in each region was than tested for correlation with the previously obtained cytokine levels in plasma using Pearson correlation.
