I have treated my breast cancer cells with 5uM 5-AZA for 3 days and and checked out the expression of the genes previously known to be methylated in breast cancer. The expressions of these known genes were induced to around 10x after 5-AZA treatment.
After that I scanned a bunch of genes and found their expression to be induced to the levels ranging from 2x to more than 1000x. However, when I sequenced the promoter of a gene which was induced more than 1000 fold in AZA treated cells, I found out that the promoter region of the gene was not de-methylated at all (even in some cell lines, the methylation seems to be increased a little).
Another gene's promoter turned out to be in the same situation. How is this possible? Please share if you have any comments or suggestions.(My bisulfite sequncing primers do not include CpGs, and the bisulfite conversion seems to be allright according to QUMA software (>95%))
Thanks in advance.