Quantifying DNA Methylation by Pyrosequencing

Bisulfite-modified DNA was amplified by PCR in a 50 μl reaction volume, using the primers described in Additional file 4 and reagents supplied by Applied Biosystems. A 40 μl aliquot of each PCR product was used to perform the pyrosequencing reaction following the manufacturer’s protocol and as previously described [26 (link)]. Negative controls recommended by the manufacturer were used, as well as positive controls that included (i) DNA in vitro methylated using SssI CpG Methyltransferase (New England Biolabs, Hitchin, UK) following the manufacturer’s instructions, (ii) hypomethylated DNA, generated through PCR and a (iii) mixture of equal volumes of the above methylated and unmethylated controls. The methylation quantification was analysed by Pyro Q-CpG Software (Biotage, Uppsala, Sweden).