Hello there Ari

We also have encountered this problem before, wherein drug selected cells still show negligible reduction of protein, even though they should be expressing the specific shRNA. Do you by any chance have a fluorescent reporter in IRES with your shRNA?

Another issues can be the cell line. Most cultured cells have deregulated pathways and may surprise you in the way they may regulate levels of certain proteins (isoform switch, etc). Also, does your target protein have a long half-life, or is it stored in reserves in some cell organelles? This may make it difficult to visualize knockdown anytime soon. If there is someway you could trigger the cells to employ that protein, maybe the cell will be pushed to transcribe more of it, and in the presence of an shRNA (given that its effective) a clear reduction of protein will also become evident possibly.

qPCR is a good idea to check transcript levels and worst case, its worth to try out siRNA mediated transient KD.

Hope this helps

Regards

Aparajita

I would agree with Aparajita's response. In addition, I would add that your protein of interest might be regulated at the translational level more than the transcriptional level. Instead of shRNA, you might try CRISPR-mediated methods of protein knockdown. We have used this with great success and numerous proteins in many different cell lines. Best of luck!

If you transfect transiently siRNA, (not a plasmid base construct), theoretically all the cells will contain it and it will work in every cells.