I have not been doing qPCR very long but hopefully you all can help me. I am working with a NFkappaB luciferase reporter and transfecting/co-transfecting into my cells. Unfortunately my mentor does not like the whole measure the lucfierase activity....i still to do this day do not understand why because this is the way most if not all scientists do. Anyways so I have been trying to look at emergence of fluoesense (luciferase) by qPCR with my normalizing factor being 18s rRNA. Honestly to me this seems a little silly because not all my cells are taking up this plasmid and normaling to this endogenous gene does not make sense to me. The NF kappa B plasmid does have hygromycin resistance gene on it. I was thinking about making primers for this and using this as my internal standard. Does this sound good to anything? Is this a silly idea? Please help---any advice would be grateful.