Quantitative pyrosequencing analysis of
methylation. (A) Methylation levels of RARβ2 were quantitatively measured using pyrosequencing. The pyrograms of RARβ2 show low levels of RARβ2 in unmethylated CpGs (upper) and high levels in methylated CpGs (lower). (B) Methylation levels of RARβ2 were compared between 62 normal control tissues from formalin-fixed paraffin-embedded tissue and 214 hypermethylated tumor tissues (P < 0.0001; Wilcoxon rank-sum test). (C) To determine if RARβ2 hypermethylation is associated with transcriptional silencing, RARβ2 mRNA levels normalized to GAPDH were compared between methylated (N = 14) and unmethylated (N = 34) fresh-frozen tissues. The fold change in RARβ2 mRNA levels was significantly different between the two groups (P < 0.0001; Wilcoxon rank-sum test).