5-AZA/TSA upregulation of gene in cell lines - Gene induction (Jul/05/2005 )
I'm wondering if others have enountered this issue. I have two cell lines, one positive, representing "normal" expression for my gene and the other cell line, negative for expresssion. I've done the preliminary theoretical CpG island prediction, subjected the genomic DNA to MspI/HpaII digestion and have correlated the subsequent PCR with expression profiles from the lines ie. PCR product with HpaII digestion but none with MspI from the negative line.
I've incubated with 5-aza (in DMSO) and TSA (in ethanol) separately and together. There is no response to the 5-aza over a wide range of concentrations but a minor one with the TSA. I'm having difficulty finding references that see this specific TSA induction with NO help from the 5-aza, even when combined.
Has anyone observed this?
there are papers out there about TSA induced demethylation do a pubmed on TSA and DNA methylation. MAny describe combination treatments as well as singluar treatments.
you may see a synergistic effect of demethylation combining TSA and 5-aza-dC.
however I think your assay my not be sensitive enough to reach this conclusion as you are testing the methylation of a CG within a seqeunce of CCGG, you may want to look in more detail with bisulfite seqeuncing.
I have done the searches on PubMed and the combination of the chemicals for the induction. I can't find and references for just TSA induced so if anyone knows of any, I'd appreciate it.
As for the sensitivity comment, can you explain that a little more?
try this link
Selker et al 1998 and here you will find what you are looking for, you can also try a search for papers citing this one.
as for your specificity. I am assuming you are performing a restriction enzyme PCR assay.
This will only test the methylation status of the restriction site within the region of interest. You can not assume methylation status of the surrounding sites just by restriction digest and then a PCR. If you have multiple restriction sites within the PCR amplicon, disgestion of one site is enough to result in no amplification, so you won't be able to tell which site was methylated or not. This was what I was on about with regard to sensitivity.