1 reply to this topic

[molbio]

Hi,

I'm quite new to AZA/TSA but have done some experiments exploring if gene expression is increased after treatment of cells with AZA and/or TSA.
Does any one have any good suggestions on what to think about when setting up experiments like this? If I don't see a difference in expression levels, how do I know that it is because I haven't treated the cells too short time or with to low concentrations? But if I increase these parameters there seems to be toxic effects...
When I do real-time PCR I also find that my house-keeping/reference genes seem to be affected. Any good suggestions for reference genes that are stable and isn't affected by AZA or TSA?

Any ideas are highly appreciated!

[methylnick]

hi molbio,

I would first look to see if anyone has published AZA/TSA treatments in the system you are using first. If you are unlucky and people have not used the drugs then you would have to perform a titration of both drugs to assay for reduction in growth. I would suggest assaying the drugs independantly to determine the optimal concentration and length of time for treatment and then combine the two together.

As for a suitable RT-PCR control. I could be worng and please correct me, but house keeping genes should not be affected by these drugs as they are always "switched on" or expressed so there should be no problem in that respect.

good luck with your studies!

Nick