siRNA Reverse Transfection-Based Gene Silencing In Vitro: A Procedure to Deliver Small Interfering RNA in Cultured Cells to Inactivate Target Gene Expression
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Pipette the siRNA with improved minimum essential medium to mix, and incubate for 5 minutes at room temperature. Add the transfection reagent and the improved minimal essential medium to the siRNA, and pipette to mix. Incubate it for 20 minutes at room temperature. Then, add the transfection mix to each well of the collagen-coated plate.
Wash the cells in the 100-millimeter Petri dish twice with DPBS. Add 5X trypsin to the cells, making sure to cover the entire surface with the trypsin. Wait for 30 seconds, and carefully remove the trypsin. Incubate the Petri dish for 5 to 10 minutes at 37 ℃ in the incubator.
Then, tap the dish to detach the cells, avoiding cell damage. Add 10 milliliters of DMEM without pyruvate, 25 mM glucose, 10% FCS, and 1% penicillin and streptomycin, to neutralize the trypsin. Carefully pipette the medium up and down to detach the cells and homogenize the cell suspension.
Count the cells using a Malassez counting chamber or an automated cell counter, and adjust the concentration of the cells to 6.25 x 105 cells/mL of medium. Seed 800 microliters of the cell suspension per well of a 12-well plate, or 400 microliters of the cell suspension per well of a 24-well plate containing the transfection mix.
Incubate the plates in a cell culture incubator, and do not disturb them for 24 hours. On the next day, carefully replace the supernatant with fresh DMEM without pyruvate, 25 mM glucose, 10% FCS, and 1% penicillin and streptomycin, and return the cells to the incubator.
