Optimization of GenMute™ reagent for siRNA silencing.

GenMute™ siRNA transfection reagent (Cat # SL100568) is one of the most potent siRNA delivery tool in the market. The optimal siRNA concentrations range from 1.0 nM to 10 nM. Excessive siRNA may lead to "flooding effect" with sub-optimal silencing. Our lab has been using GenMute™ reagent to knock down endogenously expressed growth factors with very good luck.  The following procedures will guide to optimize GenMute™ reagent for best silencing in 24-well plate. For other cell culture formats, please refer to the protocol of GenMute™ reagent.

1). Change medium and add 0.5 ml of complete medium each well of 24-wel plate (with serum and antibiotics) 30 or 60 minutes before transfection.
2). Dilute 20 pmol siRNA (5.0 µM x 4.0 µl) into 200 µl of GenMute transfection buffer (Cat # SL100572) in a sterile tube and let's sit at RT for 5 minutes.
3). Add 2.0 µl GenMute reagent, briefly vortex and keep the transfection complex at RT for 15 minutes. Please note: never keep the transfection complex longer than 25 minutes at RT.
4). Add 50 µl transfection complex to your cells directly in duplicate (final 10 nM siRNA), 25 µl complex to another 2 wells (final 5.0 nM siRNA), 12.5 µl to 3rd 2 wells (final 2.5 nM siRNA) and 6.25 µl to 4th 2 wells (final 1.25 nM siRNA).
5). Check silencing effect in the 4 different siRNA concentrations 24~48 hours post transfection and choose the best transfection conditions.