Hello everyone.

I find myself trying to do shRNA transfection on plasmid against KDM4A protein. I have trouble determining which of the three shRNAs used may have the best silencing effect on my protein.

I tried with lentivirus, but it did not go well; now I want to try in HepG2 cells using lipofectamine 3000.

How long should be the selection time of positive clones with puromycin?

I have to use Dox to induce the expression of my shRNA; what concentration would be recommended? I have read that very high concentrations can generate over-expression, affecting the results obtained. If anyone ever had this problem and was able to solve it, could you give me some tips for my trials?

I have used 2 ug/mL of Dox. Still, I have not observed any difference between Dox+ and Dox- in the qPCR results, so I was recommended to increase the concentration of Dox. Still, I think from some reports that too much shRNA expression can be wrong due to bad interactions with other cellular functions.

I appreciate your support.