Mar 30, 2018
miRNA mimic transfection with Lipofectamine RNAiMAX
- Alessandra Bisagni1,
- Stefania Croci1
- 1Azienda Unità Sanitaria Locale – IRCCS Reggio Emilia.
Protocol Citation: Alessandra Bisagni, Stefania Croci 2018. miRNA mimic transfection with Lipofectamine RNAiMAX. protocols.io https://dx.doi.org/10.17504/protocols.io.n58dg9w
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: March 30, 2018
Last Modified: March 30, 2018
Protocol Integer ID: 11168
Abstract
This protocol is provided for optimization of miRNA mimics transfection in a 6-well plate. Lipofectamine® RNAiMAX Transfection Reagent is a proprietary formulation for transfecting small RNAs (e.g., siRNA, Silencer® Select siRNA, Stealth® RNAi, mirVana™ miRNA mimics and inhibitors) into a wide range of eukaryotic cells.
Guidelines
RNA-Lipofectamine® RNAiMAX complexes must be prepared in serum-free medium such as Opti-MEM® Reduced Serum Medium and can be added directly to cells in culture medium, in the presence or absence of serum and antibiotics. It is not necessary to remove complexes or change/add medium after transfection unless toxicity by Lipofectamine® RNAiMAX is observed.
Seed 2x105 cells into a 6-wells plate with 2 mL RPMI + 10% FBS plus antibiotics (Day 0).
Plated cells has to be 60-80% confluent at the time of transfection. (Day 1).
Dilute 5.0 μL Lipofectamine® RNAiMAX Reagent in 125.0 μL Opti-MEM® Medium per each well that has to be treated.
Dilute 2.0 μL miRNA in 125.0 mL Opti-MEM® Medium per each well that has to be treated.
Add 125 μL diluted miRNA to 125 μL diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio).
Incubate both solutions for 5 minutes at room temperature.
00:05:00
Change the medium from cells to remove dead cells.
Add 1750 μL fresh medium.
Add 250 μL of relative miRNA Lipofectamine complexes to the wells as shown in figure belove.
Incubate cells for 1–3 days at 37°C. Then, analyse transfected cells.
Note: As controls it is important to include cells treated with Optimem alone, cells treated with Lipofectamine alone (to evaluate the toxicity of the transfetion reagent) and cells treated with a negative control oligonucleotide to evaluate aspecific effects.