I want to study the stability of mRNA by adding Actinomycin D on transfected cell line. What is the suitable time to treat the transfected cells with Actinomycin to inhibit RNA synthesis? Is it after 24 hrs posttransfection or more?
I did a first trial at 16 hrs posttransfection using 5 microgram/ml Actinomycin as recommended and collect the RNA at 0, 2, 4, 6 and 8 hrs. Upon doing Real time-PCR of cDNA, there was no change in the mRNA level which means that Actinomycin did not affect the RNA. Now I am thinking this is may be because I treat the cells at early stage ….. Any opinion?