Bisulfite Conversion and Pyrosequencing

Bisulfite pyrosequencing was performed as described previously (19). Briefly, 0.5 µg of genomic DNA was bisulfite-modified using the EZ DNA Methylation Kit (Zymo Research, Orange, CA, USA), according to the manufacturer’s protocol. The bisulfite-modified DNA was subjected to PCR amplification using a tailed reverse primer in combination with a biotin-labeled universal primer. PCR and sequencing primers were designed using PyroMark Assay Design 2.0 (Qiagen GmbH, Hilden, Germany). The CpG site of miR193a was PCR amplified with specific primers ( Table 3 ) in a 25 μl reaction using Invitrogen Platinum™ DNA Polymerases (Invitrogen). Prior to pyrosequencing, 1.5 μl of each PCR reaction was analyzed on 1% agarose gel. Pyrosequencing was performed on the PyroMark Q24 instrument (Qiagen) using Pyro Gold Reagents (Qiagen), according to the manufacturer’s protocol. The methylation level of 11 CpG sites, which are located +5 to +11 was measured. The methylation percentage of each cytosine was determined by dividing the fluorescence intensity of cytosines with the sum of the fluorescence intensity of cytosines and thymines at each CpG site. In vitro methylated DNA (Merck Millipore, Billerica, MA, USA) was included as a positive control for pyrosequencing.