siRNA Transfection Protocol

How to perform siRNA transfection with Lipofectamine RNAiMAX protocol. Superior siRNA/miRNA delivery and gene knockdown. In this video, we will perform an siRNA transfection experiment using Lipofectamine RNAiMAX reagent. As always, use good cell culture practices and wear your personal protective equipment. Clean the cell culture hood and work surface by spraying and wiping them down with 70% ethanol. The day prior to your transfection, seed your cells so that they will be 60% to 80% confluent at the time of your experiment. The day prior to your transfection, seed your cells so that they will be 60% to 80% confluent at the time of your experiment. For this transfection experiment you will need: - Lipofectamine RNAiMAX reagent - Opti-MEM Reduced-serum Medium - siRNAs diluted to a working concentration of 10 micromolar. We will be using two Ambion Silencer Select siRNAs, a BLOCK-iT Alexa Fluor red fluorescent siRNA, and a negative control siRNA - Five, 1.5 milliliter microcentrifuge tubes in a rack - A P200 and P10 pipette and appropriate tips - A marker and a timer - And a 24-well plate with 60% to 80% confluent cells We will be following the 24-well plate format of the Lipofectamine RNAiMAX protocol. Because we have 4 siRNAs, we will prepare a master mix of RNAiMAX. Add 200 microliters of Opti-MEM medium and 12 micoliters of RNAiMAX in a tube labelled "Master Mix" Mix well by vortexing or flicking the tube. Add 50 microliters of Opti-MEM Medium into each of 4 tubes and label them 1, 2, Positive, and Negative. Add 3 microliters of each 10 micromolar siRNA stock to its corresponding tube. Mix well. Add 50 microliters of the RNAiMAX master mix to each of the siRNA dilutions in tubes 1, 2, Positive, and Negative. Incubate the complexes for 5 minutes at room temperature. After the 5-minute incubation, remove your 24-well plate containing your cells from the incubator and bring it to the workspace in the hood. Add 50 microliters of the siRNA-reagent complex from tubes 1, 2, Positive, and Negative to wells 1 to 4 of the 24-well plate, respectively. You should have enough volume to run duplicates if desired. Place your 24-well plate back into the incubator and grow cells for 1 to 3 days at 37 Celsius. After incubating the cells 24 hours at 37 degrees Celsius, assess the transfection efficiency of the BLOCK-iT Alexa Fluor red fluorescent siRNA using the FLoid cell imaging station or microscope. To assess gene knockdown use a quantitative method such as Ambion Cells-to-Ct Kit and Real-Time PCR. For transfection protocols, FAQs, troubleshooting, and tips & tricks visit http://www.thermofisher.com/transfection