siRNA Transfection Protocol_kor

Clean the cell culture hood and work surface with 70% ethanol using good aseptic technique. Seed cells one day prior to transfection so they reach 60-80% confluence by experiment time.

Collect all required materials including Lipofectamine RNA iMax reagent, Opti-MEM medium, siRNAs diluted to 10 micromolar concentration, microcentrifuge tubes, pipettes, and the 24-well plate with confluent cells.

Add 200 microliters of Opti-MEM medium and 12 microliters of RNA iMax to a single tube labeled 'master mix' and mix thoroughly by vortexing or flicking.

Distribute 50 microliters of Opti-MEM medium into four labeled tubes (1, 2, positive, and negative), then add 3 microliters of each 10 micromolar siRNA stock to its corresponding tube and mix well.

Add 50 microliters of the RNA iMax master mix to each siRNA dilution tube and incubate all complexes for 5 minutes at room temperature.

Remove the 24-well plate from the incubator, add 50 microliters of each siRNA-reagent complex to the corresponding wells (tubes 1, 2, positive, and negative to wells 1-4), and return the plate to the incubator.

After 24 hours at 37°C, evaluate transfection efficiency of the Alexa Fluor red fluorescent siRNA using the EVOS cell imaging station or microscope to visualize red fluorescence.

Use quantitative methods such as the Ambion Cells-to-CT kit and real-time PCR with primers for your target gene and housekeeping gene to assess gene knockdown efficiency after 1-3 days of incubation.