distinguish between T and C after bisulfite sequencing - (Apr/07/2005 )
Hallo dear Colleagues!
many thanks for this forum, so many very useful tips for performing the CpG anaylsis of the promoter region...
Maybe you already discussed this problem but i could not find in any previous topic:
so, i have 4 samples (2 different controls and 2 samples treated with siRNA (where i expect different patter of methylation). Everything worked very good including sequencing, BUT: in the samples with siRNAs i can not clearly distinguish between C and T on the electropherogram after the sequencing, there are the peaks of both colours (corresponding to T and C) of the same height and on the same positions just there where i expect the changes. On both controls there are clearly CG´s. I did it now for the second time and the picture is the same. How would you explain it? what could it be?
Thanks a lot!
I assume you are doing direct sequencing, right? If yes, that's quite normal. The overlapping C&Ts mean partial methylation (some molecules are methylated at that CpG site while others not). If you want a accurate quantitation, do a TA cloning especially in the case that siRNA may not induce full methylation even at the target site.
Danke schoooeeen, pcrman...but then it could be that i pick up exactly the clone where it is methylated or the second would be not methylated, then should i pick 10 clones and just look for the majority of CG´s at these positions?? another question: could it be that from 25 CpG in the core promoter 3 are methylated/not-methylated and that´s enough for transcription regulation?
Yes, ten clones can well represent the real percentage of methylated vs unmethylated. If cost is not a problem, you can sequence more.
It is hard to predict exactly which CpG methylation or how much CpG methylation may silence the gene. Maybe the more methylated CpGs, the less transcription. In my experience, siRNA induced methylation won't spread much out the target site at least in human cells.