Ez dna methylation gold kit 200

DNA isolation was performed using a DNA isolation kit (Qiagen, QIAamp DNA Mini kit 50). The methylation status of promoter regions of P15, P16, RB1, and MGMT was determined by methylation-specific polymerase chain reaction. Therefore, 500 ng DNA of each tumor specimen as well as appropriate control samples were treated with bisulfite (Zymo Research, EZ DNA Methylation-Gold kit 200) (33 (link)). In summary, unmethylated cytosine was converted to uracil, whereas methylated cytosine remained unchanged. The modified DNA was recovered by ethanol precipitation and suspended in polymerase chain reaction (PCR) grade water. For analyzing the methylation status, the primer sequences listed in Table I were used (34 (link)–36 (link)). PCR was performed using a 25-µl reaction volume and 38 PCR cycles. All PCR products were electrophoretically separated on a 2% agarose gel. As a positive control, a chemically globally methylated DNA was used (Zymo Research, bisulfite-converted Human DNA). Genomic DNA isolated from a non-neoplastic dura mater tissue served as a negative control. In addition, each PCR included a control without any DNA template. An example of PCR results is presented in Fig. 1.