Transfection in Reverse
Ambion was acquired by Applied Biosystems in 2006 and is now a division of Life Technologies. The Ambion brand encompasses a range of products for isolation, detection, quantification, amplification, and characterization of RNA, including its siPORT NeoFX® neotransfection agent, a lipid-based formulation that the company claims can be used to efficiently transfect adherent cells as they are subcultured without increased cytotoxicity
According to Ambion, low transfection efficiencies as well as poor cell viability are the most frequent causes of failed gene transfection studies and that use of reverse transfection, or neofection, reduces failure rates.
Michel Cannieux, product manager, Ambion RNA interference technologies, has found that reverse transfection technology addresses the problem of getting reproducible transfection efficiencies from “well to well, and tube to tube.”
Traditional transfection requires that cells be passed 24 hours prior to transfection. “In traditional transfection techniques, on the day of transfection, complexed siRNAs, transfection reagent, and often a transfection aid are added to the wells in a relatively small volume, to the larger volume of culture medium containing the cells. The addition of a small volume to a larger one results in inefficient mixing, particularly in the case of 96- to 384-well plates.”
As a result of poor mixing “some cells receive a larger dose of transfection reagent, leading to localized toxicity and transfection inefficiencies within each well.” By contrast Cannieux says that, in reverse transfection the siRNA and transfection reagent contained in just a few microliters are distributed first into the wells. The cells in a 10–20 larger volume of culture medium are overlaid onto the mixture in the wells while the cells are still in suspension.
Cannieux says that while efficient transfection is crucial for the outcome of any RNAi experiment, with chemically synthesized siRNAs in particular, “you don’t have the luxury of being able to eliminate nontransfected cells as you would using a selection marker on a plasmid vector. High-transfection efficiency and a robust and reliable protocol that works for most cell lines is important and become paramount for siRNA screening programs. Transfection efficiency has to be comparable from plate to plate, for the whole set of siRNAs tested, sometimes several thousands, as in the case of genome-wide screens.”