I have tried to treat mouse ileum/colon tissue with Actinomycin D in an organ bath up to 12 hr time points.  I used 18s as my housekeeping gene.  When I isolated RNA with Trizol I got a very high concentration of RNA using nanodrop and the purity looked fine.  When I did RT-PCR I saw a step wise fold reduction in the 18s Ct value however for my gene of interest I did not see a reduction in Ct value.  This leads to a relative expression that is increasing overtime rather than decreasing.  Can anyone tell me what could be the possible reason for this? I am thinking that since I see a reduction in 18s Ct value the actinomycin D treatment is working.