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Split cells after siRNA transfection? - What to do when cells become confluent after transfection of siRNA (Oct/30/2006 )


For siRNA transfection, most protocol recommends transfection at >40% cell density and transfect for 72-96hrs to get the best knock-down effect in protein level.

If the doubling time of the cell line is around 24hrs, 2 days after transfection, the cells would for sure be confluent. Shall I let the cells stay there for another 1-2 days or split the cells to lower density?

It seems overgrowth will decrease knock-down efficiency (in my hands, not 100% sure).

However, if I split the cells, would a large percentage of transfected cells be killed during splitting? Would a second transfeciton required?

I know I should do the experiments to figure it out. But I do hope I could get suggestions from people who have done similar experiments.



I'm having the exact same question as you. I need to kill my silenced cell with okadaic acid, but when they are 100% confluent, the cells don't absorb the toxin as well as when they are 80-90%


In my experience (using human melanoma cell lines) , the siRNA effect is lost once you change the medium, e.g. when you split the cells. Why dont you split them and retransfect? There is also the possibility to add transfection mix when you seed the split cells (reverse transfection or sth like that). Or just use less cells in the beginning.
Hope it helps,



Sorry, but the optimal cell density for maximum transfection efficiency varies with each different cell line, you just have to determine it empirically. The critical factor are 1] how large the cells are and 2] how fast they grow. Also, if the cells are 'sticky' and clump up when you trysinise them for plating you'll get poor transfection efficiency. Plus, if you try transfecting at very low density you'll probably see nonspecific toxicity from the transfection reagent.

I usually try to have my cells at 20-25% confluency for transfection, but this varys up or down depending on how big they are/ fast they grow. For the ~25 different cell lines I've done transfection with, I aim to have the cells ~75-80% confluent 48 hours post-transfection - if the siRNA is going to work, you should certainly see very good knock-down of protein by this time-point [70%+]. I've even transfected quiescent cells and gotten ~50% knockdown of my target protein by 48 hours post-transfection [tho of course the stability of the protein you're trying to silence has a large impact on this].

As a jump-off point I would recommend trying a plating density of somewhere between 1-1.5x 10^5 cells per well [of a 6-well plate, use less cells if they are really large], and harvest the cells for analysis at 24, 48 and 72 hours - if you get good silencing at 48 hours [and the cells aren't completely confluent] probably no point in pushing for 72-96 hours.


I agree with Hooly. You should check when you have knockdown: I get a very good knockdown even at 24 hours and I don't see any difference between 24 and 48 hours (of course it depends on the gene I'm silencing and the siRNA I'm using)


i agree with Hooly and dnafactory. We would transfect cells when they r 25-40% confluent and get more than 80-90% down regulation in 24 hrs. Dont go by what the supplier recommends. try it out for urself and optimise ur experiment accordingly.


I agree with the comments from all three above. Just do not use the same amount of transfection complex. You need to use less lipids and DNA when you transfect less cells or you will kill cells and get false knockdown results.