I am carrying out transfection of HEK293 cells using Lipofectamine 2000. In a 96 well plate I am co-transfecting a pGL4 CRE luciferase vector along with my pRL-Tk Renilla control vector (50 ng firefly: 5 ng Renilla). Normally, I'll carry out the transfection for 4-6 hours in Optimem media and then replace with HEK media and leave my cells in the incubator overnight. The next day I stimulate with forskolin for 6 hours then analyze luciferase activity using the Dual Luciferase reporter assay. The firefly signal looks fine but the Renilla signal is barely detected. I am using a SpectraMax M3 to take my luminescence readings. I have tried boosting my Renilla quantity to 25-50 ng but still don't get a decent signal.
Any tips/suggestions would be appreciated.
In our case, after transfection (electroporation with Neon system and dual reporter pDluc plasmid) we wait 24 hrs, then replace HEK medium into a HEK medium with stimulator of translational readthrough (aminoglycosides). After next 24 hrs of incubation we measure luciferase activities using the Dual Luciferase reporter assay (Promega). The level of both luciferases is satisfying. Maybe you should try longer incubation with forskolin?
Dear Sushanth,
check this link out:
https://fi.promega.com/resources/product-guides-and-selectors/protocols-and-applications-guide/transfection/
In our case, after transfection (electroporation with Neon system and dual reporter pDluc plasmid) we wait 24 hrs, then replace HEK medium into a HEK medium with stimulator of translational readthrough (aminoglycosides). After next 24 hrs of incubation we measure luciferase activities using the Dual Luciferase reporter assay (Promega). The level of both luciferases is satisfying. Maybe you should try longer incubation with forskolin?
Thank you for the replies. It could just be that my cells are getting too old (~P24) and just take longer to express Renilla. I was wondering if anyone else had observed this same phenomenon. Renilla should give me a constitutive, basal expression regardless of forskolin.
Longer time for the Renilla to express- try different concentrations of Renilla after allowing at least 36hr after transfection. Why would passage number be important if you are getting results with the other reagent?
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