Hello everyone! I am currently investigating re-expression of a tumor suppressor gene in breast cancer cells, namely MDA-MB-231 cells, by 5-Aza-2′-deoxycytidine treatment.
I have tried various concentrations & durations of Aza treatment but for some reason I don't see really significant changes in methylation. I am using pyrosequencing to investigate methylation levels within the promoter of the gene.
I started with 5 umol/l Aza all the way up to 10 umol/l for 72h (with daily medium changes) but still no changes in methylation. Interestingly, I do see changes in mRNA expression (up to 116-fold).
Aza is stored at 80°C in DMSO aliquots and only thawed immediately before usage.
Do I need to use even higher concentrations of Aza? Or can certain CpG sites be "immune" to demethylation by Aza?
Thanks!
Hi,
I am not sure about the reason but you may think about:
- 5´-Aza effect on methylation is strongest in proliferating cells. Maybe an increased treatment time will increase the number of cells which are affected
- some cell lines are quite sensitive to 5´-Aza and show increased cell death at concentrations >5µM. Have you observed increased cell death or diminished proliferation?
- are other genes affected and show a demethylation?
Best,
Norman
Dear Lars,
1. Short answer - perhaps you need to combine your Aza-Treatment with Histone-Modification reagents (like TSA) to see the DNA methylation modification - provided - effect.
2. Now, long answer - your readout is PCR. There are plenty of factors involved: binding of TF to the promoter, binding the co-factors to the promoter, availability of DNA promoter to the TF, etc. Perhaps Aza-Treatment is just not enough to see the changes. Here is the expert in DNA Methylation- Pavel Bashtyrkov-just drop him a message, he´ll be happy to look at it precisely
Best Luck!
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