Demethylation by 5-AZADC treatment - (May/26/2008 )

Hi all,

Does someone know how to use 5-aza-2-deoxycytidine to treat NIH3T3 cells for demethylation? What is the concerntration of 5-aza-2-deoxycytidine I should use for NIH3T3 cells? And how many days I should treat in order to get demethylation?

Thanks!

hi Zebra,

there is no easy way to answer this without caveats.

The best thing to do is to perform a titration with 5-azadC and a time course with a readout of cell viability to determine the toxicity of 5-azadC on your cells.

This is best because your NIH3T3 most certainly will behave differently to those of another lab, this would be due to freeze/thawing, passage number and that they may not be the cell line you thought (I won't go there). There would be many papers that have published with 3T3 cells and this would give you a guide, but you need to optimise with your system.

Good luck.

Thank you very much Nick.
I will try to perform the titration.

The person who pioneered 5-aza-dC is Peter Jones. Look up his papers from the 80's to 90's. The condition for good treatment is also pH dependent. The titration is a good idea. There will be some stock to stock variation so you want to make one large stock and test it to calibrate.

You want to grow your cells when they are in log phase, then when you change your media, change to one that has fresh aza. Only replicating cells will incorporate aza into DNA. The demethylation rate depends on how efficient the aza is getting taken up. Aza causes the Dnmt1 to covalently linked to DNA while trying to methylate, but then stalls. This causes the level of dnmt1 to drop and as a result, demetylation occurs passively over time. This takes days, and depends on the cell type and locus question. Remember, if your cells stop growing because you have too much aza, aza won't incorporate no matter what. So your cells need to be replicating and dividing.