Hi Melissa. I always have issues with stable knockdowns. Plasmid or viral-based strategies for shRNA delivery lead normally to random integration in the cell's genome. This can cause positional effects (mutations in the already screwed-up genome of most cultured cell lines) as well as leading to different levels of shRNA expression depending on the area of integration. If you really want stable shRNA, I would suggest an inducible system where you can test the biological effect in the presence or absence  of shRNA expression in the SAME cell line/population. My bottom line is that if I was to trust a result, I would have much more confidence with siRNA when compared to shRNA. This is from personal experience, I have been using siRNA with cultured cells from the early days, 2001...

Hi Melissa. I always have issues with stable knockdowns. Plasmid or viral-based strategies for shRNA delivery lead normally to random integration in the cell's genome. This can cause positional effects (mutations in the already screwed-up genome of most cultured cell lines) as well as leading to different levels of shRNA expression depending on the area of integration. If you really want stable shRNA, I would suggest an inducible system where you can test the biological effect in the presence or absence  of shRNA expression in the SAME cell line/population. My bottom line is that if I was to trust a result, I would have much more confidence with siRNA when compared to shRNA. This is from personal experience, I have been using siRNA with cultured cells from the early days, 2001...

Melissa, 

I agree with Miguel, we have used both transient knockdowns using electroporation of shRNA and lentivirus based stable shRNAs. Depending on your shRNA your cells might start to compensate by increasing or reducing the expression of other signaling proteins which will affect your signaling assays. It could also be that the protein being suppressed longterm may intrinsically control the genomic expression of other proteins. Since you have already used two different shRNAs the latter is more likely to be true.

Another question is do you also get 90% knockdown in your transient siRNA based transfections? knockdown efficiency which is likely higher in the stable cells may give you alternate results. Stables are always convenient and are my first choice, but if you are having problems you can try inserting your shRNA into lentiviruses carrying inducible shRNA promoters (pLKO.1) or use transient knockdown (electroporation of plasmid shRNA, or siRNA)

Hi Melissa, just to add something else for comments above. As Miguel said, for sure that insertion can induce problems. However, you did not mention how you make stable cell lines, whether you transfect shRNA plasmid and you do selection until you have plasmid integration into genome or you are infecting with retro/lenti viruses, because what will be inserted into genome could be different being much better by viruses (the insertion's problem is same for both, but by viruses you are sure that full cassette is inserted, plasmid integration could be only selection marker inserted without having shRNA). Since you are comparing always against scramble shRNA, if it is the case that insertion is causing troubles you should see insertion troubles with scramble too (sometimes it is better compare scramble against mock cells, should be same expression levels). Besides, you could do more than one infection in parallel to make sure have same results. A question, when you confirm KD, Are you performing WB? or by RT-qPCR?. Last comment, since siRNA normally is transfected in high concentration you could induce immune activation, toxicity, etc, on the other hand, shRNA produce siRNA but since it is made by cellular machinery its concentration is much lower, inducing less toxicity than transfected siRNA...this could be an issue if those effect are involved in the pathway that you are studying...Hope that it helps!

Like it was said before in the ShRNA assay the genome integration is random; thus, that's why you can have several different levels of suppression of your target protein. 

Another problem of this assay is the off-target effects; however, it is easy to avoid this. You should use at least three constructs with different ShRNA sequences and a blast negative sequence (control). To affirm that in your assay there's no off-target effect you have to reproduce the same effects in all three constructs sequences. 

I've worked with ShRNA and they can be tricky, but it is a very good tool if properly executed can give incredible results. 

I hope it helps. 

Hi Leonardo,

do you like to share your protocol which gave you good results for the shRNA transfection? I had also more success with siRNA transfection so far.

I followed the protocol on Addgene website for pLKO.1 but used Lipofectamine 2000 for transfection and I did in 10 cm plates so I used about 60 ul of Lipofectamine. I used 24 ug of total DNA using the second generation packaging (remember it is BSL2+) and the viruses are pantropic.

If you need more help with that or have specific questions you can call me. If you need help with stable cell line generation I can help you provided I am included on the authorship.

Best wishes

Abid Kazi PhD