Lipid siRNA transfection

For some cell lines, either electroporation or cationic lipids may be used. Basically any tfx protocol will require you to work out conditions for every cell line. Sometimes between 70-90% or more cells can be transfected with cationic lipids. With a bit of tweaking, and depending on the cell line, you can drive siRNAs into almost 100% of the cells with the following protocol:

Seed cells high, so that they are 85%+ on the day of transfection:

For one 10 cm dish:

40 ul Lipofectamine 2000

20 ug DNA

100-300 pmoles siRNA

tube 1

add the siRNA and/or DNA to 2 ml SF-DMEM (no drugs added)

tube 2

add the lipid to 2 ml SF-DMEM (no drugs added)

mix these two tubes immediately, for a volume of 4 ml, and then add to cells which have been washed 2X with SF-DMEM. Incubate 3-6 hours, then add normal media. Check for expression or knockdown 36+ hours post-transfection. Knockdown by siRNAs usually last 3-5 cell doublings.

When oligofectamine is used, the identical protocol is used, but:

Seed cells at 15% confluency, perform tfx with 30 ul oligofectamine and siRNAs, then repeat tfx 36 hours later. Analysis is performed when cells are 85-90% confluent. I have not calculated the transfection efficiency with oligofectamine.