Lipofectamine™ 2000 Reagent is a lipid-derived reagent designed to deliver exogenic nucleic acids (DNA and/or RNA) into eukaryotic cells, either adherent or in suspension. Thee two most important factors in choosing transfecting reagent are: high efficiency and low cytotoxicity. According to the manufacturer, and my own experience (using human embryonic kidney cells, HEK 293), Lipofectamine™ 2000 fulfills both of these criteria.
Lipofectamine™ 2000 is very easy to use. A specific, precalculated amount of the reagent needs to be complexed with DNA/RNA in Opti-MEM medium. Next, the real transfection starts by adding these complexes to the cells and allowing them to adhere to the cell surface and deliver their cargo into the cells. This step can take from 30 minutes up to a few hours. I would recommend, at least at the beginning, to monitor the transfection under the microscope. You can observe that initially cells round up their cell bodies and processes, but later on, their morphology goes back to normal: cells adhere tightly to the bottom of the dish and extend processes. It is also important that transfection takes place in the presence of serum, which helps to minimize the transfection stress and avoid major changes in cell physiology, often triggered by serum removal.
It is also very important to optimize transfection conditions. Too much Lipofectamine™ will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. What is nice about Invitrogen’s Lipofectamine™ 2000 is that it has an extensive, very useful manual which addresses these issues and helps tremendously cut down the initial time of protocol optimization. I would start with the smallest amount of Lipofectamine™ recommended (0.5 uL per ug of DNA), which should be the least cytotoxic. The manual recommends also that a certain amount of nucleic acid, described in ug, should be used for transfection. I would calculate ug into moles and try to estimate the ratio between the number of plasmid molecules and the number of cells in order to best understand the ratio giving optimal transfection efficiency. These calculations are of particular importance, when performing co-transfections (e.g. simultaneous transfection with plasmid DNA and with miRNA precursors). For example, miRNA precursors should be in molar excess over mRNA derived from plasmid cDNA to sufficiently inhibit translation.
On the downside, transfection needs to be performed in medium without antibiotics, which is somewhat inconvenient as you need to prepare antibiotic-free media especially for the transfection. After transfection, I routinely remove the medium containing the remaining Lipofectamine™/nucleic acid complexes - you can called it old habit (acquired when using older transfecting reagents), but I just don’t like the idea of lipids (which can impair cell membrane integrity) hanging around if it is not necessary, even if their cytotoxicity is low. According to the manufacturer, this step is not necessary due to low cytotoxicity of the reagent, which surely makes the use of Lipofectamine™ 2000 even more convenient.
Another advantage of Lipofectamine™ 2000 is its application for co-transfection experiments in which plasmid DNA and siRNA are used simultaneously. I actually have used it very successfully when cotransfecting plasmid DNA with miRNA precursors. I have never observed any disadvantages of using two different nucleic acids instead of one, and co-transfection gave very good results.
Lipofectamine™ can be highly recommended. It was optimized to work in many mammalian cell lines, where it shows high efficiency and low toxicity, and can be a transfecting reagent of choice for the growing demand of DNA/siRNA co-transfections.
Instructor
Department of Psychiatry
University of Massachusetts