Pyrosequencing analysis of bisulfite converted DNA

Genomic DNA was extracted using a genomic DNA purification kit (Qiagen Company, Hilden, Germany). Bisulfite modification of DNA was performed by the EZ DNA Modification Kit (Zymo Company, New York, USA) according to the manufacturer’s protocol. Bisulfite-treated DNA was PCR amplified using SPAYS2 specific primers. Primers were designed using Pyrosequencing Assay Design Software v2.0 (Qiagen, Germany). Subsequently, the PCR product (10 uL) and magnetic beads (3 uL) were pipetted into the PyroMark Q48 disk wells and loaded on the Q48 instrument (Qiagen, Germany) for PSQ. Amplification was conducted according to guidelines suggested by Pyrosequencing Assay Design Software. The methylation level was analyzed using the Q48 Autoprep software 2.4.2 on CpG analysis mode and visualized as sequence-specific programs.