With shRNA-expressing vectors, it is always a good idea to include a fluorescent marker (e.g. GFP; there is plenty of space, as the shRNA is small!), so that you can assess the transduction efficiency of your virus. If the viral titre has decayed over time in storage, this will show as weaker fluorescence or fewer fluorescent cells.

If you are talking about separate viral preps between your experiments, then it is expected that there will be differences in the amount of virus you get from prep to prep. Are you quantifying the amount of virus you are getting, making sure you are adding the same amount each time?

Finally, if all of the above have been accounted for, maybe you should look into the quality/health/possible altered physiology of the cells receiving the virus. Also, different cell types may require different amounts of virus for the same effect.

As for your second question, that really depends on the protein/gene you are studying. Sometimes even a small decrease is enough for an effect (after all, there are - for example - diseases that are due to heterozygous loss of a gene, so 50% reduction). In other cases even a 90% knockdown may give a different phenotype than a true homozygous knockout. And there is, of course, everything inbetween!

I hope this helps!

I'm working with shRNA-mediated silenced cells and have seen a range of knockdown efficiency primarily dependent on the cell line used. However, a higher selection pressure right at the offset may help.

Hi Ioannis and Sam. Thank you so much! This really helps. I appreciate your insight.