siRNA Reverse Transfection-Based Gene Silencing In Vitro: A Procedure to Deliver Small Interfering RNA in Cultured Cells to Inactivate Target Gene Expression

1. Preparation of precoated plates

1. On the day before or a few hours before the transfection, prepare a solution of collagen type I at 100 µg/mL in 30% ethanol from a stock solution at 1 mg/mL. Add 250 µL of collagen per well of a 12-well plate and 125 µL per well of a 24-well plate, and spread the solution over the surface of the well.
2. Leave the plate without the lid under the culture hood until the collagen dries. Wash twice with Dulbecco's phosphate-buffered saline (D-PBS).
   ​NOTE: Precoated plates are available for purchase.

2. Preparation of the transfection mix

NOTE: The final concentration of siRNA is between 1 and 100 nM (1 to 100 pmol of siRNA per well of a 12-well plate). The final concentration of the miR mimic is 10 nM (10 pmol/well). Determine the best concentration of each siRNA, miR mimic, or other oligonucleotide prior to starting the experiment to avoid off-target effects. Perform transfection experiments in triplicate to facilitate statistical analysis of the results. Prepare all reagents in excess to account for normal loss during pipetting.

1. Mix by pipetting (volume/volume) the siRNA (or other oligonucleotides) with improved Minimal Essential Medium (Table 1). Incubate for 5 min at room temperature.
2. Add the transfection reagent and the improved Minimal Essential Medium to the siRNA, and pipet to mix (Table 1). Incubate for 20 min at room temperature (during this time, proceed to section 3). Add the transfection mix to each well of the collagen-coated plate.

3. Preparation of the 3T3-L1 adipocytes

1. Wash the cells in the 100 mm Petri dish twice with D-PBS. Add 5x trypsin to the cells (1 mL per 100 mm dish), making sure to cover all of the surface with the trypsin. Wait for 30 s and carefully remove the trypsin.
2. Incubate the Petri dish for 5-10 min at 37 °C in the incubator. Tap the 100 mm dish to detach the cells.
3. Add 10 ml of DMEM without pyruvate, 25 mM glucose, 10% FCS, and 1% penicillin and streptomycin to neutralize the trypsin. Carefully pipet the medium up and down to detach the cells and homogenize the cell suspension.
4. 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 µL of the cell suspension/well of a 12-well plate (5 x 10⁵ cells) or 400 µl of the cell suspension/well of a 24-well plate (2.5 x 10⁵ cells) containing the transfection mix.
   NOTE: One 100 mm Petri dish of adipocytes will allow the preparation of one 12-well plate or one 24-well plate. A 100 mm dish usually contains 6-7 x 10⁶ adipocytes, which correspond to 5 x 10⁵ adipocytes per well of a 12-well plate.
5. Incubate the plates in a cell culture incubator (7% CO2 and 37 °C), and do not disturb the cells for 24 h. On the next day, carefully replace the supernatant with fresh DMEM without pyruvate, 25 mM glucose, 10% FCS, and 1% penicillin and streptomycin.
   ​NOTE: It is also possible to seed the cells into collagen-precoated 48- and 96-well plates but take more precautions when replacing the media to avoid detachment of the adipocytes.

Table 1: Transfection reagents required for 12-well and 24-well plate formats.