co-transfecting DNA and RNA duplexes - (Jan/26/2006 )

I am trying to evaluate wether a transcriptional co-repressor is involved with the activity of a particular transcription factor. I have developed a system that relies on reporter gene expression from a transfected plasmid. I would like to use RNA duplexes to knock down endogenous co-repressor and eveluate the effect this has on the expression of my reporter gene. Does anyone have experience with tranfecting both DNA plasmids and RNA duplexes into cells? Should I co-transfect or perform sequential transfections? To make matters worse these are difficult to transfect primary cells. Any help would be greatly appreciated.

Hi,

I have done some DNA and siRNA cotransfections. I don't transfect them together because it is hard to predict the transfection efficiency for each type of molecule. So I first transfect my reporter plasmid in a 100 mm dish. After 3-6hrs, I harvest the transfected cells and plate them into 6-well plate, the second day I tranfect siRNA into the cells. This works pretty well.

There is a specific reagent from QIAGEN jsut for that purpose. Check it out.

http://www1.qiagen.com/Products/Transfecti...ionReagent.aspx

Hi,

I was wondering how long you wait to harvest cells - after transfecting with siRNA? If possible, I dont want to wait more than 48 hours after the first transfection (with plasmid) but then 24 hours might not be enough for sirna ..?

I am glad I came across this discussion because i was looking for some tips on how to handle such cotransfections.

Thanks,
S