I have transfected Hep G2 cell with my promoter of interest having pGL3 backbone and the pRL-TK vector as internal control using lipofectamine ltx following the company protocol. After 24 h performed dual luc assay (Promega). I used the TK vector as 20:1 ratio (promoter of interest: TK vector). After 24 h of incubation I stimulated the cells with a cytokine for 1 h. And I got the following luminometer reading?

Sample                                                  RLU (firefly)                   RLU (Renilla)

1 cells without transfection                            107                                    304

2 only TK vector                                            131                                   7009

3 promoter of interest & TK vector                35122                            458000  

4 promoter of interest & TK & cytokine         43143                             905007

Now my  questions are-

- Why is the renilla signal so different in sample 2,3 & 4, although the volume of TK is same in all samples?

- May there be any effect of TK on the cytokine I applied for stimulation?

- Is there any effect of the size of promoter of interest with the transfection efficiency?

- Is there any effect of the volume of the TK vector for the transfection of the promoter of interest?

- Is there any effect of cell number on the transfection efficiency?

- what would be the reasons for these abnormalities?

I tried the assay before with increased cell number, the result as follows-

Sample                                                  RLU (firefly)                   RLU (Renilla)

1 cells without transfection                           127                                   203

2 only TK vector                                            143                                 100598

3 promoter of interest & TK vector                25301                            658097  

4 promoter of interest & TK & cytokine         5034                             350346

I cannot take any decision from these kind of results. 

Please share your valuable suggestion and related paper (if any).

Thank you