% confluency question - when to transfect the cells? (Jun/16/2008 )

Hi everyone,

I have a questionon the confluency % question. I am planning to use Lipofectamine 2000 for 2 different experiments. 1. transfecting siRNA alone and 2. cotransfecting siRNA with a plasmid.

According to the protocol, for siRNA alone transfection 30-50% confluency is recommended. For cotransfection 90-95% is recommended.

I am not sure that I understand why there is a need for difference in confluency? As the transfected DNA and RNA are not lethal, if the cells are 90-95% confluent at the time of transfection, at 24h after transfection, they are going to be crowded, growing on top of each other and losing their shape. Right? Wouldn't it be ideal to transfect at ~50-60% confluency for both?

I would appreciate your replies.

Thanks,
Micron

Hi everyone,

I have a questionon the confluency % question. I am planning to use Lipofectamine 2000 for 2 different experiments. 1. transfecting siRNA alone and 2. cotransfecting siRNA with a plasmid.

According to the protocol, for siRNA alone transfection 30-50% confluency is recommended. For cotransfection 90-95% is recommended.

I am not sure that I understand why there is a need for difference in confluency? As the transfected DNA and RNA are not lethal, if the cells are 90-95% confluent at the time of transfection, at 24h after transfection, they are going to be crowded, growing on top of each other and losing their shape. Right? Wouldn't it be ideal to transfect at ~50-60% confluency for both?

I would appreciate your replies.

Thanks,
Micron

I routinely transfect a 6-well plate at 60-80% confluency and then move them to a bigger dish the next morning. Co-transfecting with hairpin, with and without plasmid, and GFP-histone gets me anywhere between 50-90% transfection efficiency depending on my cells' temperaments.