This is our protocol for reverse-transfection of siRNA in 96- or 384-well plates. The efficiency of gene silencing is at least as good as with a forward transfection protocol (in which you first plate cells and let them adhere and grow overnight before the transfection). This protocol is however much simpler, and can be automated on the Beckman Biomek FX or the Agilent Bravo. Importantly, it allows the preparation of ready-to-use plates that can be stored at -80ºC. Whenever a screen is carried out, all that needs to be done is thawing the appropriate plates and dispensing cells in each well using the BioTek EL 406. Washing the cells is not necessary. Transfection mix can be present for the full three days of incubation.

1.Prepare the siRNA mix.

For 96 well plates: 5µl of siRNA (1 µM) + 5 µl of Optimem per well. For 384 well plates: 2.5 µl of siRNA (1 µM) + 2.5 µl of Optimem per well. Mix and wait 10 min.

2.Prepare the carrier mix.

For 96 well plates: 0.1 µl of Lipofectamine2000 + 9.9 µl of Optimem per well. For 384 well plates: 0.05 µl of Lipofectamine2000+ 4.95 µl of Optimem per well. Mix and wait 10 min.

3.Prepare the transfection mix. 

Mix the carrier mix with the siRNA mix. For 96 well plates: 10 µl of carrier mix + 10 µl of siRNA mix per well. For 384 well plates: 5 µl of carrier mix + 5 µl of siRNA mix per well. Mix and wait 15-20 min.

4.Add the transfection mix to the wells. 

For 96 well plates: 20 µl per well. For 384 well plates: 10 µl per well. At this point, plates can be stored at -80ºC for at least 3 years without loosing transfection efficiency.

5.Plate the cells.

For 96 well plates: ~2500 cells in 80 µl of cell suspension per well. For 384 wells plates: ~1000 cells in 40 µl of cell suspension per well.

6. Let the cells grow for 3 days.