Apr 16, 2024
siRNA Transfection of Dispersed Islet Cells
This protocol is a draft, published without a DOI.
- 1University of Alberta
Protocol Citation: Amanda Gomes, Xiong Liu, Xiaoqing Dai 2024. siRNA Transfection of Dispersed Islet Cells. protocols.io https://protocols.io/view/sirna-transfection-of-dispersed-islet-cells-czbnx2me
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 30, 2023
Last Modified: April 16, 2024
Protocol Integer ID: 87118
Abstract
siRNA Transfection of Dispersed Islet Cells
Materials
Silencer‱ FAM-labeled Negative Control No. 1 siRNA
Catalog number: AM4620 (ThermoFisher)
Opti-MEM‱ I Reduced Serum Medium
Catalog number: 31985070 (Gibco)
Lipofectamine‱ RNAiMAX Transfection Reagent
Catalog number: 13778075 (ThermoFisher)
Accell Non-targeting Control Pool
Catalog ID: D-001910-10-05
siRNA of your target gene
Before start
Reconstitute your siRNA to a 20 μM working solution with nuclease-free water.
Work in a cell culture hood, in an RNAse-free environment, and keep the hood light off.
Clean the cell culture hood, turn the UV light on, and thaw reagents on ice.
Disperse islets, and resuspend cells in Opti-Mem media after centrifugation. Distribute 100 μL of media with cells per 35 mm dish.
Remember to make extra dishes for qPCR to test the efficiency of the transfection.
For each control dish (calculate the volume of reagents according to the number of dishes you wish to make):
Tube 1: 50 μL OptiMem media + 3 μL Lipofectamine RNAiMAX Transfection Reagent
Tube 2: 50 μL OptiMem media + 0.6 μL control siRNA + 0.4 μL dye indicator (Silencer‱ FAM-labeled Negative Control)
For each dish with the target gene silenced:
Tube 3: 50 μL OptiMem media + 3 μL Lipofectamine RNAiMAX Transfection Reagent
Tube 4: 50 μL OptiMem media + 0.6 μL target siRNA + 0.4 μL dye indicator (Silencer‱ FAM-labeled Negative Control)
Wait 5 minutes after making the 4 tubes.
Mix tubes 1 and 2, and tubes 3 and 4. Wait 15 minutes.
Add 103 μL of transfection reagents to each dish with 100 μL of media with dispersed cells.
Wait 4-6h, and add 2 ml of human media (DMEM) to the dishes.
After 2-3 days, perform desired experiment and add 0.5 -1 mL of Trizol to the extra dishes. Freeze cells with Trizol in the -80°C freezer until RNA extraction.