Hello, hard to answer without knowing the details (construction, targeting, etc etc), but RNA expression does not necessarily equal protein expression. When there is RNA and no protein expression, the most straightforward reason is that something is out of frame somewhere. Of course, this is the simplest, and an examination of the locus of interest may give more insight as well.

Not very helpful, I'm afraid,, but if you are worried about GFP expression in HepG2 cells (which should be find as far as I know), you can do a quick transfection of one of your controls (the actual GFP fusion of your protein of interest) and see if you get green (again, depends on your fusion, etc etc), and at the very least, just use plain old GFP.

before doing higly sofisticated techniques first check if your cells are fluorescent (even in a transient transfection) if not then check the sequence of your plasmids...

Hello, probably you might need to check back the Plasmid which was used to construct the GFP to find out whether it has any tags (for example His/Flag). If there is a tag, then probably after overexpressing or knockdown, check the protein expression of the respective tag. Probably you can also perform FACS analysis to check whether there is a GFP signal expression after transfection along with both positive and negative controls.

Hi Eglantine. Obviously a number of things could be amiss. Not sure what you mean by "good" expression. HepG2 are not particularly adept (vs HEK293T) at expression high levels of recombinant proteins; ddPCR can be quite sensitive; how many copies of transcripts per cell are you getting?. I would perform a control transient transfection in HEK293T or another easy to transfect cell line (HepG2 can be temperamental). If you don't detect your GPF within 24 h of transfection, your home made construct is likely at fault. So long as your construct contains a strong promoter (what's your promoter?) sequence, detection in HEKs should be easy.

Thank you all very much for taking the time to share your thoughts.

Nicolas Kuperwasser my construct isn't a "classical" GFP fuse to a protein of interest. The GFP itself is my protein of interest to report the activity of a Cis sequence.

My plasmids are constructed as followed:

Cis sequence of interest / a miniCMV promoter / GFP protein

Nicolas Kuperwasser and Kousalya Lavudi Yes a frame shift would explain my issue but I have 4 different contructs and I can't detect GFP in any of them which make me think the problem may be elsewhere.

Kousalya Lavudi thank you for your suggestion, I will definitely check on tags.

Didier Poncet the sequencing of the amplified and purified plasmid and the sequencing of the gDNA of transfected cells align perfectly with the initial plasmid sequence. The issue must be elsewhere.

Sébastien Soubeyrand thank you for your suggestion, I will definitely test my plasmids in HEK cells.

GFP expression can be quite different among clones, it varies between 300 to 15000 eGFP copies per ng of cDNA. mRNA doesn't equal protein expression for sure but with these levels of expression, I should be able to see some GFP wouldn't I ?

My plasmids are actually commercials ones (Genecopoeia) , they've been custom made but not by me, I left it to the experts.

Thanks again everyone for your insights.

I'm happy to take further advice and suggestion.

All the best