I am currently doing methylation specific PCR and I find that all my samples are showing bands for both methylation and unmethylation in both the control and tumor samples. I wonder is it because of partial bisulphite conversion?
Real Sumayya Abdul Sattar
It is very common and because of the simple fact that not all the cells in the tumor samples are tumor cells. Another reason is intratumor heterogeneity, i.e not all the cells will have the same methylation levels within the tumor cells from the tumor sample.
Solution: use the gel intensity as a semiquantitative measure of methylation levels/ do it on real time macchine for better quantification.
What is your control sample? If you are using blood (or any other sample that you think it should be unmethylated), and you get bands for both methylated and unmethylated status it is highly probable that your Bisulfite transformation is the culprit. If your control sample is adjacent "normal" tissue, the problem may be something similar to what Mohit mentioned.
Be sure to include controls for both fully methylated and fully unmethylated status.
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