Stepwise analysis of 32 selected genes in breast cancer. (A) Cell line analysis confirmed frequent methylation (>30% of samples) in cancer cell lines but not normal/control breast tissue in 16 genes (highlighted in white), which were selected for further analysis. Thirteen genes (highlighted in light grey) did not show methylation in the region analysed by our CoBRA primers and were removed from further analysis. Three genes (highlighted in dark grey) showed frequent methylation in cancer cell lines but also normal breast tissue and were removed from further analysis. (B) Analysis of the 16 genes found to be frequently methylated in cell lines were analysed in primary breast cancer patient samples. Frequent methylation (>25% of samples) was observed in nine of these genes (highlighted in white) and were carried forward for expression analysis.