It is highly unlikely.

Even if the sequence you're amplifying through PCR is still transcribed from the knockout gene, the level of transcription of the overexpressed one should be higher.

I distinguish 4 main critical points to check:

- presence of the premature stop codon

- promoter for the overexpression

- amplification specificity

- normalization

Hi,

You need to design the qPCR primers spanning the putative mutation site, or, alternatively one pair before and one after the mutation. Like this you can really judge if there is a premature stop codon. I think that if you have the write primers you should be able to distinguish between a knockout and an overexpressor. There is a database from Roche with qPCR primes that are working. I have never had an issue with them so far. It gives you many options all over the gene of interest and you can simply choose the region you want. It is still working although they will discontinue it: https://lifescience.roche.com/en_de/brands/universal-probe-library.html#assay-design-center

I agree with Nicolò M. Villa and Svetlana Boycheva Woltering