Dear Laura,

Overexpressing a gene using a pcDNA plasmid is a transient transfection. Thus, it basically depends on that when you measured the mRNA level of your cells (how much time was passed from your initial transfection). It is also noteworthy that typically the half-life of proteins are longer than mRNAs. Thus, it is possible that the protein product remain a while after degradation of their corresponding mRNA.

Question: how confident are you that you're actually measuring any transgene at all?

I would recommend you design (and test) primers that allow you to distinguish endogenous transcripts from transgene-derived transcripts: if you've added a tag to your transgene (flag, myc etc) this should give you some transgene-specific sequence to target.

This will allow you to determine the respective kinetics of transgene and endogenous transcripts, because right now one simple interpretation is that you're producing plasmid-derived transcripts and translating them to protein, but you're just not measuring the mRNA (measuring the -unchanged- endogenous levels only).

Also, you don't list any sort of time course for this: how soon after transfection do you measure gene expression? How soon do you measure protein?

I think you can still get a high level of protein expression without mRNA (degraded) what I don't know if it corresponds to this gene.

A high expression of proteins does not necessarily mean you have mRNA, you may get high copies of mRNA which could a high level of protein expression but you might also get one copy of mRNA where there is a high level of protein expression. Not necessarily the level of mRNA need to correspond to protein expression. You may get a high expression of genes with no change in mRNA level. And in the same way you may get a high level of protein expression with a high gene expression.

(You know you have a high level of protein expression, but you are not so sure about the mRNA?)

Thanks John Hildyard for your answer. It was very helpful!

I use Luhmes cells which is a dopaminergic neuronal precursor cell line which can be differentiated in 5 days.

I've transfected the cells both at day 2 of differentiation (measuring the mRNA at day 5), but also at day 5 of differentiation (measuring the following day, at day6).

There is no tag to my transgene in the plasmid I'm currently using, but I do have primers against other areas of the plasmid. I will give them a try.

Thanks again!