Treatment with AZA/TSA - (Aug/27/2004 )

Hi,

I'm thinking about treating cell lines with 5-Aza-2'-deoxycytidine and Trichostatin A and then do expression analyzes on extracted RNA. The reason is I'm getting tired of doing primer optimization for bisulfite sequencing/MSP. The problem is that I can't find any protocol for this, I've looked in articles but it's hard to figure out what they've really done. Concentrations and times used seem to vary.
Do any one have a protocol? What do you think about my strategi?
Also, how should I handle these chemicals, I guess they aren't that good for you...

Thanks in advance,

I'm looking forward to any suggestions!

-molbio-

Hi,
that's what I found somewhere on website...
Will this help?

Treatment of cells with 5-aza-2 -deoxycytidine and/or trichostatin A

Human glioma cell lines were plated at a density of 5 10 cells/dish. After 24 h, 1 M of 5-aza-2 -deoxycytidine (5-aza-dC) (Sigma) or the same volume of ethanol as a control was added to the medium. The culture medium was changed every two days. Five days after starting the treatment of 5-aza-dC or ethanol, 5-aza-dC with or without 100 ng/ml of trichostatin A (TSA) (Wako Pure Chemicals, Osaka, Japan) were added to the medium. All the cells were harvested and analyzed after 24 h.

PS: I have no reference to this info. And (unfortunately) I have no personal experience with this.

-Paja-

QUOTE

The problem is that I can't find any protocol for this, I've looked in articles but it's hard to figure out what they've really done. Concentrations and times used seem to vary

Do you mean protocol for cell treatment with aza or TSA? No, there is no universally applied protocol for this. A appropriate protocol has to be customized for each cell type which varies dramatically in their tolerance to the drugs.

Basically there are two types of protocols for treatment, one is consecutive treatment, the other non-consecutive. It all depends on how sensitive your cells are to these drugs. Before starting your experiment, you have to determine a dose-response curve. If your cells are very sensitive, try non-consecutive treatment and low doses.

Dissolve aza in PBS or culture medium, TSA in ethanol. They are not very toxic to your body.

-twister-

Thank you for your replies. I guess I have to try, starting with the dose-response curve. What do you mean by consecutive and non-consecutive treatment? Should I add new AZA/TSA every time I change medium?

Thanks again!

-molbio-

consecutive means treating cells continously without washing out. if your cells are sensitive, treat your cell nonsecutively, for example, plate cells at day 0, add drug at day 1, 3, 5, change medium (without drug) at day 2, 4, 6, or you can treat continously at a lower dose.

-twister-