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

Overview

In this video, we describe a procedure to perform reverse transfection of siRNA into adipocytes to modulate the expression of a target protein.

Protocol

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.

	Per well (12-well plate)	Per well (24-well plate)
Oligonucleotides	20 µL	10 µL
(si-RNA, miR…)
Improved Minimal Essential Medium	20 µL	10 µL
Transfection reagent	5.6 µL	2.8 µL
Improved Minimal Essential Medium	154.4 µL	77.2 µL
Total volume of transfection mix	200 µL	100 µL

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

Materials

Name	Company	Catalog Number	Comments
12 well Tissue Culture Plate	Dutscher	353043
2.5% Trypsin (10x)	Gibco	15090-046	Diluted to 5x with D-PBS
2-Propanol	Sigma	I9516
3-Isobutyl-1-methylxanthine	Sigma-Aldrich	D5879
Bovine Serum Albumin (BSA)	Sigma	A7030
Collagen type I from calf skin	Sigma-Aldrich	C8919
Dexamethasone	Sigma-Aldrich	D1756
D-PBS	Gibco	14190144
Dulbecco's Modified Eagles's Medium (DMEM)	Gibco	41965062	4.5 g/L D-Glucose; L-Glutamine; no Pyruvate
Ethanol	Sigma	51976
miRIDIAN micro-RNA mimics	Horizon Discovery
FAM-labeled Negative Control siRNA	Invitrogen	AM4620
Newborn Calf Serum	Gibco	16010-159
Fetal Bovine Serum	Gibco	10270-106
Penicillin and Streptomycin	Gibco	15140-122
FAM-labeled Negative Control siRNA	Invitrogen	AM4620
ON-TARGETplus Mouse Plin1 siRNA SMARTpool	Horizon Discovery	L-056623-01-0005
Free Glycerol Reagent	Sigma-Aldrich	F6428
Petri dish 100 mm x 20 mm	Dutscher	353003
Transfection reagent (INTERFERin)	Polyplus	409-10
Improved Minimal Essential Medium (Opti-MEM)	Gibco	31985-047
Insulin, Human Recombinant	Gibco	12585-014