2.4 Bisulfite conversion and CpG pyrosequencing analysis
The methylation status of single CpG sites along the studied sequence was analysed by the Pyrosequencing CpG assay methodology. Bisulfite conversion reactions for each DNA sample conducted prior to Pyrosequencing for the complete conversion of the unmethylated Cytosines (existing as CpG dinucleotides) to Uraciles, with Epitect Bisulfite Kit (Qiagen).
Sequencing and PCR primers were designed with the PyroMark Assay Design Software version 2.0 (Qiagen) (Table 2).
TABLE 2: Pyrosequencing primer sets