I used the pGL4.2 vector to construct my target promoter vector and co-transfected it with an independent beta-gal vector as an internal control. I also transfected two different transcription factors (TFs), A and B, to assess their effects on the target promoter.

In HEK293T cells, I co-transfected the plasmids using the T-Pro reagent. TF B is 2000 bp larger than TF A. After 24 hours, the beta-gal absorbance results showed that B TF had much higher values than both A TF and the empty pGL4.2 vector. Additionally, the luciferase signal was several times higher for B compared to A.

However, in cancer cells, where I transfected the same plasmids, the beta-gal internal control values were similar across all conditions, while the luciferase signal for B TF was only twice that of A TF.

My questions:

1. How should I interpret these results? Which one should I trust?

2. Does the size of a co-transfected plasmid affect the transfection efficiency of other plasmids?

3. HEK293T cells do not express the target protein, but cancer cells do. Could this affect the results?

Would you like me to refine any part of it further?