I assume that the cells contain a mingenome which is your Gaussia reporter and that you expect an increased signal after successful transfection. Cotransfection of four plasmids and an additional RNA is quite ambitious. If nothing works you have to perform several control experiments. Check your transfection efficiency by substituting one of your plasmids with an EGFP expression plasmid. To get efficient coexpression you need an efficient transfection.

Control whether the proteins are expressed by Western blotting.

I would also suggest to discuss your problems with experts using your MARV system.

Uwe Borgmeyer Exactly, upon successful transfection, I am supposed to have an increase signal in the dish having the minigenome and the support plasmids.

I just read a paper where a different ratio has been used. plus the paper demonstrated that three support plasmids (L, NP and VP35) are enough for vRNA transcription and replication... I currently am subculturing some new cells to repeat the experiment. This time around, I will a new ratio (NP/VP35/L(/VP30) : 1/5/5(/1)) and the experimental design below

* first dish: vRNA minigenome

* second dish: vRNA + three plasmids (NP/VP35/L)

* third dish: vRNA and four plasmids (NP/VP35/L/VP30)

I also tough of checking the expression of those proteins by Western Blotting (but, i dont have the antibodies yet)

Thank you again for your suggestion

Best

Steve