Before and after demthylation - Difference in meth pattern? (Feb/04/2008 )
can anybody give me a reference where they compared the methylation profile of a promoter before and after demethylation, preferably using snager sequencing or pyrosequencing? I don't believe I have come across one yet and I'd like to see what the actual difference is.
in this case i would do the classical Bisulfite Sequencing. Meaning
Bisulfite Treatment , PCR , sucloning and sequencing.
Pyrosequencing would only make sense if you have your own Pyrosequencer :-)
Sorry, maybe I should make myself a bit more clear. I have used both techniques to determine meth profiles of several genes which were hypermethylated vs controls but after demethylation I can't see a real difference between the profiles, even though I get massive induction of the mRNA.
I've also encountered this recently - there is a big increase in gene expression but there is not even a slight change in the methylation of the CpG island. I don't have a useful reference for you however. Perhaps something along these lines hasn't been published because it goes against the theory that a hypermethylated promoter can be associated with active transcription?
this result is not surprising.
this could be due to histone modifications at your prmoter so you are not seeing in DNA methylation or the methylation of something upstream of your gene of interest (transcription factor that activates the transcription of your gene for example) .