Methylation Analysis of miR-210 Promoter

From each sample, 500 ng genomic DNA was subjected to sodium bisulfite modification using the EZ DNA Methylation-Gold™ Kit (Zymo Research, Irvine, CA, USA) and eluted in a final volume of 110 μl H₂O according to the manufacturer's instructions. Methylation assessment of Region 1, 2 and 3 of the miR-210 promoter was performed by MS-HRM analysis and the primers were designed to amplify both methylated and unmethylated DNA [60 (link)–62 (link)]. PCR and HRM were carried out on a LightCycler^® 480 (Roche, Mannheim, Germany) with each 10 μl reaction comprising 2 × MeltDoctor^TM HRM Master Mix (Life Technologies, Carlsbad, CA, USA), 3 mM MgCl₂, approximately 10 ng bisulfite modified DNA and 500 nM of each primer. The methylation assessment of each region was performed by melting profile comparison between each sample and DNA methylation standards derived through a serial dilution of 100% methylated DNA (Universal Methylated Human DNA Standard, Zymo Research, Irvine, CA USA) into 0% methylated DNA (EpiTect Control DNA, Qiagen, Hilden, Germany). All analyses were done in triplicates and the technical specifications for the Region 1, 2 and 3 MS-HRM assays, including primer sequences, genomic location, assay-specific PCR cycling and HRM protocols are listed in Supplementary Methods.