Standard curve constructed for RASSF promoter methylation. The dilutions of methylated DNA in unmethylated DNA are as follows: 0% methylation, 10% methylation, 50% methylation, and 100% methylation. Using Meth Primer software, a pair of primers was designed to amplify both methylated and unmethylated sequences after bisulfite conversion. The melting curves of 0% and 100% methylation indicate melting temperature of unmethylated sequence (76.6°C) and methylated sequence (81°C).