siRNA Reverse Transfection-Based Gene Silencing In Vitro: A Procedure to Deliver Small Interfering RNA in Cultured Cells to Inactivate Target Gene Expression
Published: April 30, 2023
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 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. Leave the plate without the lid under the culture hood until the collagen dries. Wash twice with Dulbecco's phosphate-buffered sal…
Divulgations
The authors have nothing to disclose.
Materials
| 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 |
Tags
Citer Cet Article
siRNA Reverse Transfection-Based Gene Silencing In Vitro: A Procedure to Deliver Small Interfering RNA in Cultured Cells to Inactivate Target Gene Expression. J. Vis. Exp. (Pending Publication), e20978, doi: (2023).