Figure 3

Qualitative analysis of CST6 methylation by MSP. (A) Locations of oligonucleotide primers for methylation-specific PCR (MSP) and pyrosequencing are shown relative to the transcriptional start site within the human CST6 gene. "Pyro-" represents pyrosequencing. (B) The sensitivity and specificity of the MSP primers were verified by amplifying 100% methylated DNA and 100% unmethylated DNA with sodium bisulfite treatment. (C) MSP for the CST6 gene was performed using methylation-specific (M) and unmethylation-specific (U) primer sets, respectively, in formalin-fixed paraffin-embedded tissues from IBC and DCIS. The numbers shown are sample identification numbers. "Pos" and "Neg" represents positive and negative controls for methylated (M) and unmethylated (U) allele, respectively. (D) No relationship was found between CST6 methylation by MSP and the cystatin M loss in DCIS (P = 0.38) and IBC (P = 0.50).