Hi

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.

Best Regards
Michael

I would say talk to the company whose product you are using. You did not mention the company name. it is hard to say

saswati1 said:

I would say talk to the company whose product you are using. You did not mention the company name. it is hard to say

Hi Saswati1

This is the background:
I use a PGL3 plasmid which contains a luc gene under control of a cloned promoter region which is induced by adding certain drugs. When I cotransfect this reporter with expression plasmids encoding certain proteins I see a suppression of the luc reporter. Having done this in a 1:1 ratio (e.g. 250ng/250ng in 500ng total) I observe a 50% suppression of the luc activity.

My inquiry is not related to the luc reporter that I use. I have done control transfections with internal luc reporters from PROMEGA together with my particular experimental reporters from the same company to test for differences in transfection efficiency, cell viability and effects of certain drugs that I use to induce cells (and reporters) with. However, my question is which approach to take to monitor a stronger effect than I already see in my preliminary experiments.

And this is what I actually want to know:

Should I take into account the molar ratio of my two plasmids. The reporter containing the promoter region is smaller (approx. 5kb) than the expression plasmid (approx. 7.5kb), and if I add more of the latter to compensate for the difference in size (which affects how many copies of each plasmid that are taken up by cells and eventually encoded into protein) I probably get a higher suppression of the luc reporter. Or, should I use another approach to investigate how many molecules of suppressive factor are needed for complete (relatively) suppression of luc activity of my particular reporter?

Sincerely
Michael