Methylation assay

To examine the methylation status of galanin, we performed methylation-specific PCR (MSP) using bisulfite-treated DNA from ten gastric cancer cell lines. DNA denaturation and bisulfite conversion were carried out using an EZ DNA methylation kit (Zymo Research, Foster City, CA, USA) according to the manufacturer’s instruction. The bisulfite-treated DNA was amplified with methylation-specific primers and unmethylation-specific primers as described in Table 1. We performed bisulfite sequencing PCR (BSP) to confirm the results of the methylation assay using the primers that we designed using MethPrimer (http://www.urogene.org/cgi-bin/methprimer/) [17]. For Sanger sequencing, PCR products were purified using a PCR purification kit (Qiagen, Redwood City, CA, USA) and then cloned into pCR2.1 TOPO vector (Invitrogen). At least ten clones were sequenced using T7 and M13 primers. We used universal methylated DNA (Chemicon International Inc., Temecula, CA, USA) as a positive control for MSP and BSP. Genomic DNA extracted from peripheral blood lymphocyte (PBLC) was used as a control.