siRNA Transfection Protocol

Clean the cell culture hood and work surface with 70% ethanol. Seed cells the day prior so they reach 60-80% confluence by the time of transfection.

Gather all required materials including lipofectamine RNA IMAX, Optimum medium, siRNA stocks, microcentrifuge tubes, and pipettes. Prepare a master mix by combining 200 µL Optimum medium with 12 µL RNA IMAX in a labeled tube and mix well.

Add 50 µL of Optimum medium into each of four labeled tubes (1, 2, positive, and negative). Add 3 µL of each 10 µM siRNA stock to its corresponding tube and mix well.

Add 50 µL of the RNA IMAX master mix to each of the siRNA dilutions in tubes 1, 2, positive, and negative. Incubate the complexes for 5 minutes at room temperature.

Remove the 24-well plate with cells from the incubator. Add 50 µL of the siRNA-reagent complex from each tube to wells 1-4 of the plate respectively. Return the plate to the incubator.

Grow cells for 1-3 days at 37°C. After 24 hours, assess transfection efficiency of the blockade Alexa Fluor red fluorescent siRNA using the Floid cell imaging station or microscope.

Use a quantitative method such as AMBION CELLS to CT kit and real-time PCR to assess gene knockdown. Prepare primers for your target gene and housekeeping gene for analysis.