I keep seeing everywhere that we have to use optimem for the lipofectamine to take the miRNA into the nanovesicles. But why? How could FBS be jeopardizing this process? Also, when the complex nanovesicle-miRNA is formed, does having FBS in the media for the cells about to be transfected affect the transfection? What are the biochemical processes involved between the FBS and the lipofectamine?
It is believed that known (albumin) or unknown proteins form complexes with lipofectamine which hinder their efficiency.
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However, lipofectamine itself exerts toxicity which affects cell growth and that impacts efficiency, that is another issue. If not optimem, then you can go for low serum concentration
Dear Pablo,
As Saurabh suggested, the most effective reagent for RNAis is RNAimax. I just would like to add that after forming of nanovesicles, the efficiency, in case of Lipofectamine3000, varies according the type of cell. In other words, for some cell FBS 20% achieves the highest efficiency and for others is vice versa. So you have to optimize that according to your experiment itself.
Best ...
Hello Pablo Llevenes
Lipofectamine transfection reagent is a cationic-lipid transfection reagent that relies on cationic lipids to facilitate nuclei acid delivery into cells. The cationic lipids form a unilamellar liposomal structure with a positive surface charge and fuse with nucleic acids to form a transfection complex. By charged interactions between the cationic lipid headgroups with the negatively charged cell membrane, the transfection complex localizes to the cell surface for endocytosis and transfection.
Serum contains albumin which negatively interacts with the lipofectamine limiting their ability to interact with cellular membrane receptors. Albumin in FBS will block the aggregation necessary for your reagent with the nucleic acid to sediment and the positive charge on these particles necessary to enter the cells, thereby hindering their efficiency.
Use of Opti‐MEM which is a serum‐reduced media contains insulin, transferrin, hypoxanthine, thymidine, and trace elements that will help compensate for the adverse impact of FBS supplementation reduction with no change in the growth rate or morphology, and at the same time will help to increase the transfection efficiency. Also, absence of serum will help to avoid possible contamination with RNases.
Best.
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