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

Abstract

Source: Jager, J. et al.  An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function. J. Vis. Exp. (2021).

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 ....

Materials

List of materials used in this article

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