for better, you should optimize your siRNA concentration using different concentration and also lipo and RNA ratio

You're mentioning you don't observe the same phenotype using a smart-pool in comparison to single siRNAs - i assume you observe the same phenotype when you compare the the three single sequences, or are you using them combined? Is, based on mRNA levels, the kd efficiancy comparable when you use the smart pool? Make sure you use the same total concentration of each siRNA, if you want to ompare directly. By using ON TARGETplus smart-pool siRNAs (with the corresponding non-targeting control pool), i usually observe a knockdown efficiancy of around 70-80 % while using a final concentration of 20nM. But that might depend on your target gene, so I'd also recommend a titration of different siRNA concentrations, until you see a comparable kd efficiancy with your previous experiments - and then hopefully the same phenotype. BTW, by using Lipofectamine RNAiMAX for siRNA transfections, i was able to significantly increase kd efficiancy by using the same siRNA concentrations as with other transfection reagents efore.

Hello Sascha Seidel,

Thank you for your answer! :)

Yep, I observe the same phenotype when I compare the the three single sequences. the smart pool is a combination of this 3 sequences +1 other

I didn't test my KD on every target because it was too much (45 diferents).

I did it with my SMART POOL, trying few concentrations and a diferent volume of Lipofectamin.

( just to know, did you test the KD by Western blot or by qPCR? and is that right to do that by qPCR?)

But for 100nM ( to have 25nM each) my cells get bad with this big amount of siRNA. So the best concentration for the moment is 50nM and my cells sems to be well

This week I planned a lot of experiments with 50 nM ( Its look like to be ok ... we will see!)

Thank you

Amandine Pic , I usually check kd efficiancy by both, WB and qPCR. The above mentioned 70-80% refer to mRNA levels, though. But i usually cross check by doing a WB, as i observed reasonable kd efficiancies at mRNA level, whereas protein levels were barely affected. My standard concentration is 20nM for the pool (5nm each) and that concentration works for most of the genes in my case. I usually do not increase the concentration to more than 60nM max, as i also observe reduced cell viability.