5-AZA, Reversing DNA methylation (epigenetic reactivation experiments) - (Jul/29/2009 )
Dear All,
I would like to perform demethylation experiments with 5 AZA on my cancer cell lines, Can anyone help me with a good protocol and suggestions. Any nice citations will be appreciated aswell.
Regards,
RKA
I found this article to be both good and easy to extrapolate a method from the method part.
Chiurazzi, P., et al., Synergistic effect of histone hyperacetylation and DNA demethylation in the reactivation of the FMR1 gene. Hum. Mol. Genet. 8, 2317-2323, (1999)
Else the articles referred to at SIGMA-ALDRICH for the 2′-Deoxy-5-azacytidine product are all quite good.
Hi RKA,
Generally there are two types of protocols: consecutive and non-consecutive treatment. The latter is to treat the cell every other day and then wash out because aza-cytidine is very toxic and some cells are very sensitive to it especially long-term treatment which is sometimes required in order to completely demethylate. The optimal concentration of azaC and length of treatment has to be experimentally determined on your cells. So there is no universial protocol for doing this.
pcrman on Aug 1 2009, 08:15 AM said:
Generally there are two types of protocols: consecutive and non-consecutive treatment. The latter is to treat the cell every other day and then wash out because aza-cytidine is very toxic and some cells are very sensitive to it especially long-term treatment which is sometimes required in order to completely demethylate. The optimal concentration of azaC and length of treatment has to be experimentally determined on your cells. So there is no universial protocol for doing this.
Thank pcrman..