Hi, I generated a stable cell line with a shRNA. However, when I tested the optimal dose of DOX to silence the target gene I obtained a working concentration at 12ug/mL... So, as the majority of all the publications have a working dose at (more or less) 1 ug/mL , I am worry that 12ug/ml is too high...
It could be possible this DOX concentration? Can I have any toxic issue related with this DOX dose that invalidate my results?
Thank you!
Doxycycline itself can affect cells, so I would be hesitant to use that high of a concentration.
When you did your dose testing, were you using the same concentrations on a non-targeting or parental cell line as a control comparison for your shRNA-expressing cells? It could be that the higher doses of doxycycline are affecting the expression of your gene of interest in addition to the silencing by your shRNA.
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