I am about to repeat a luciferase reporter experiment and need some advice concerning transfection.
In our experimental set up different luciferase reporter plasmids where cotransfected with expression plasmids encoding factors affecting the activity of the cotransfected luc reporter in subsequent measurements.
Our results showed a 50% suppression of luc activity in presence of the different expression plasmids. The experiments were performed with a 1:1 ratio of luciferase to expression plasmid.
Thus, my question concerns the experimental set up of this experiment.
What ratio of expression plasmid to experimental plasmid would be good to continue with, that is; are there any good rules to follow or do I need to empirically determine the suppressive level of the luc reporter by a certain cotransfected factor in my particular system?
As far as I understand, there are multiple strategies which can be chosen i.e.
(i) Transfecting cells taking into account the molar ratios between experimental and expression plasmid.
(ii) 2-4x excess of experimental plasmid.
(iii) Performing a dose response experiment where the concentration of expression is increased until maximal suppression of luciferase activity is observed.
I only have a limited time for this project so I wonder if anyone has any references (excluding the plethora of experimental reports) or ideas about how to proceed.