Hello,

My suggestions would be these:

- Transfection- should be done in antibiotic-free media and changed after 4 to 24 hours. The lowest confluency I've tested was 30%, any lower was counter-productive because the toxic effects of the reagent were high, in slowing growth and ultimately because you must change media and if it's too low then most of your population will not be transfected (ideally do it 40-50%). So here I would try to test different amounts of transfection reagent and time windows of exposure to the transfection mix or switch to a different one, I'm using SAINT-sRNA from Synvolux with very good results (exposure for 24h)

- Effect of siRNA is usually between 24h-48h and protein depletion after 48h or longer, but this is highly dependent on the rate of protein turnover. So an easy way would be to test your protein expression after 24h, 48h, 72h and 96h of treatment. In my case p.e. I had over 70% protein knockdown after 48h and after 4-5 days it started to reapear, because the siRNA gets degraded . Once you find the right amount of transfection reagent that wont induce cell death in your scramble (negative control) you can try to increase siRNA amount for your target to improve efficiency of KD. But sometimes efficiency just is what it is, because of your protein stability. So in summary - I wouldn't focus on cell seeding but optimizing the protocol.

- Doing transfection twice is not recommended, specially in such a short time window. It's best maybe to reconsider shRNA if you need long-term experiments or if your protein is too stable for siRNA to have a good KD.

Hope it helps ;)

I used to perform silencing experiments on mouse primary hepatocytes using RNAiMAX as transfection reagents. After transfecting the cells with siRNA. I would remove the transfection media after 4 hours. This approach allowed me to avoid excessive exposure of the hepatocytes to RNAiMAX. Subsequently, I left the cells in the plate for 24 to 48 hours to allow for effective gene silencing.

I prefer to silence cells on the bigger plates and then divide them on smaller plates for downstream treatments. This also ensures that the "same" cells are used for all experiments in one biological replicate as silencing them individually doesn't guarantee that efficiency would be the same in all the wells. This also ensures an equal cell count between siCTRL and siTARGET in the treatments themselves.

I also use RNAiMAX. I do not change the medium or use the medium without P/S. I see no difference between doing this or not. You also need to take into consideration that reagent and siRNA concentrations don't scale down linearly from bigger plates to smaller plates so your concentrations could be too high. Did you check for silencing efficiency?

So I went and did some experiments, and I found the toxicity came from Lipofectamine RNAiMAX. The general suggested protocol of 0.3 ul/well (for 96 wells) is way too high for HeLa cells, about 0.1 ul is about right (mRNA knockdown was the same between 0.3 ~ 0.1 ul, at about ~90% for my siRNA)