I'm using a Dual Luciferase Reporter Assay (firefly+renilla; Promega kit) to investigate the effect of a drug on a gene of interest. The promoter of this gene has been cloned upstream of the firefly one in a pGL4.10[luc] vector. This promoter is supposed to be activated by another transcriptional factor which is present in another vector. Just to be more clear, once seeded HEK 293T cells, the day after I do a transient transfection with pGL4.10[luc] vector containing the gene of interest; the vector containing the transcription factor and Renilla. The day after I treat cells with the drug dissolved in ethanol and wait other 24 hours. After these hours I lysate cells and do the luciferase.

I'm also using different controls, like vehicle alone and cell only cultured in media.

The problem is that I'm having very different luciferase results each time...does anyone know which could be the reason and how to try to overcome the problem?