How many fold change make sense during aza treatment - gene expression after aza expression (Dec/20/2005 )
I did an aza treatment experiment (5uM and 72h) and quantitate the mRNA expression of several genes. Two question:
1) which house keeping genes are not regulated by methylation, so are good for internal control for qPCR.
2) After aza treatment, How many fold change of gene expression can make a conclusion that a specific gene is regulated by methylation status? In my data, some gene increases dramatically such as 10-100 fold, where some change small (3 fold). can I say those genes with 3 fold or less change are regulated by methylation too.
change of exprssion by istelf after aza-dc treatment is not enough to say your gene is regulated by methylation as it could be a transcription factor or an "upstream" factor that may have been affected by the drug and it's methylation was altered that in turn affected the transcription of your gene(s) of interest, to definitively say that your genes are regulated by methylatioon you need to assay for meethylation at the promoters of your genes. be it MSP or BSP.
Actually what you said is true in my case, although the gene expression was changed dramatically in one of my genes, I can not find a CpG island in its promoter, which make me wonder two things: non-CpG island CpG sites or upstream effect. I traced two cascades upstream, still no CpG island in those regulatory factors. This make me think the non-CpG island CpG sites are the only reseason. I know there is a lot of skepticism about this issue in this forum. But I can not find any other reasons fo far. please indicate if any. Thanks CMF
there is a lot of skeptism in the field of DNA methylation that non-CpG island methylation can control gene regulation, in fact I am a supporter to this very fact and I am trying to find more evidence for such a phenomenon. Our paper looks like it is finally going to be published and it is about this very thing, I call these CpG islets, ie: regions with the same GC-content as a CpG island but are smaller. Remember MeCP2 and MBD proteins involved in chromatin structure and indirectly gene transcription, have been demonstrated to bind single methylated CpG's!!!!!!!
good luck on your quest!