Background
Reverse transfection, as the name implies, is a form of transfection conducted in the opposite manner; instead of adding nucleic acids to cells, cells themselves are added to pre-plated nucleic acids. More specifically, while forward transfection is usually used when the cells need to be attached and in their growth phase before the transfection reagent mixture is applied, reverse transfection assembles the transfection complex in the culture plate before the cells are seeded. As a bit of background, transfection is a method to transfer genetic material into cells in order to add or alter the expression of a specific gene in a cell. Transfection experiments are conducted in order to study gene expression, cell processes, DNA and RNA structure, function, and coding sequences. Transfection is typically performed using transfection reagents to transfer plasmid DNA, siRNA or miRNA into a cell.
Transfection reactions may be accomplished through the use of commercially available reagents, as well as mechanical methods of introducing DNA into eukaryotic cells. Reverse transfection is defined as the process of adding the encapsulated cargo molecule (plasmid DNA, siRNA, miRNA) while simultaneously plating the cells.
In practical terms, reverse transfection refers to cell-based array experiments in which cells are pipetted onto pre-plated cargo molecules. Such approaches are often used for large scale RNAi screening studies, where libraries of cargo molecules are on a slide, dish, or plate. The array is then incubated with cell cultures such that the cells become transfected. One of the most common applications utilizing array-based reverse transfection is large scale siRNA (or large scale microRNA) library screening using siRNA reverse transfection. Success or failure of reverse transfection depends on quality of cells, experience of the researcher, and methods used in the reverse transfection.
The micro-array based reverse transfection method was invented in 2001 by Junald Ziauddin and David M. Sabatini. Using gelatin and nucleotides (DNA or RNA), the mixture is printed onto a non-porous surface, such as glass. After drying, cultured cells are added on top of the printed nucleotides, limiting inherent variation in handling large amounts of oligonucleotides and enabling high throughput screening experiments to be performed on the same type of cells.
High-Throughput Applications
One of the primary advantages of reverse-transfection is the precision involved in getting compounds into cells. Screenings generally require an even distribution in vitro and reverse transfection, when done accurately, nearly guarantees stable quantities of transfection reagents at desired locations. The ease of actually transferring cells onto prepared plates also means that the method is easy to use for larger-scale applications, where the quantity of cells transfected is not as important as the results gained from the experiment.
In high-throughput screenings, one of the most important factors to consider is the efficiency of a given technique. Most transfection experiments require individually-tailored transfection reagents for transfecting cells, but in context of larger-scale screenings for particular cell types (such as siRNA screening), one reagent should be enough for a variety of tests to take place. Individual transfection experiments conducted in well plates, if done by hand, require an attention to precision that is prone to error. Reverse transfection, however, can limit the effects of human error as the actual distribution of transfection reagents is generally more even in the process. As a result, larger-scale transfection experiments (such as the evaluation of protein production) generally don’t require reverse transfection, but high-throughput screens may be conducted more efficiently using the technique.
A study developed a novel solid-phase reverse transfection method that would enable large biomolecules to be delivered into cells while preserving their functions. This version of reverse transfection was shown to be applicable for gene editing with CRISPR-Cas9 and antibody-mediated inhibition of protein function. Scientists found that solid-phase reverse transfection with antibodies also disrupts the function of its intracellular target when looking at anti-KIF11 antibodies. Antibody transfection enables researchers to validate gene knockouts and reduce efforts, costs, and time. The overall reverse transfection advantage comes into play by making distribution and standardization easier across different laboratories (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5630038/)