One way for researchers to deduce a gene products’ function is to eliminate that gene, hence eliminating the protein, in an organism and observe the resulting phenotype. Traditionally this has been a complicated process, but recent innovations in products for RNA interference (RNAi) have made it much simpler. It has been known for some time from studies in plants,
Drosophila, and C. elegans that short double-stranded RNA (dsRNA) oligonucleotides can silence the expression of a gene that is homologous to one of the strands within the duplex. Although the mechanisms for RNAi remain unknown, evidence suggests that dsRNA act in concert with cellular proteins to target their homologous mRNA strands for cleavage, thereby removing them from availability for translation. More recently, gene silencing by short-length small interfering RNA (siRNA) duplexes has been demonstrated to be successful in mammalian cell culture, thus facilitating the studies of specific genes and protein functions in mammals. Researchers who wish to use RNAi techniques in mammalian cell culture would often synthesize several siRNA oligonucleotides that span distinct key targets within their gene of interest, since successful RNAi of gene expression is highly dependent on the selected targets. The success rate of RNAi is also dependent on transfection efficiency, or how well the dsRNA oligonucleotides are delivered into the cells. One particular reagent that I have found useful for consistently high transfection efficiency of dsRNA into mammalian cells is the Mirus TransIT-TKO siRNA Tranfection Reagent.
The TransIT-TKO siRNA transfection reagent is reported to have a proprietary polymer/lipid formulation that, according to Mirus, should induce less cellular damage than the cationic-liposome based transfection reagents. The reagent has also been tested succcessfully for RNAi in several commonly used cell lines such as the COS-7, HEK 293, HeLa, HepG2, MCF7, NIH3T3, and Neuro-2a cell lines. Depending on the cell line being used, there are two slightly different transfection protocols, but both are easy to follow and involve little hands-on time. Incubation time of TransIT-TKO reagent/siRNA complex mixtures can vary greatly, and one therefore needs to assay for optimal conditions for inhibition of target gene expression.
Needless to say, there are many methods and reagents that can deliver dsRNA oligonucleotides into mammalian cells. There are reports that calcium phosphate-based transfection protocols do not to work for dsRNA delivery into cells while other popular and commonly used liposome-based transfection reagents (that you may already have in the laboratory) have been reported to work well. One should also remember that transfection efficiency of siRNAs is also dependent on many factors, including cell density at transfection, length of incubation time, concentration and quality of siRNAs used, and the specific region being targeted by the siRNAs. Thus, using this product or any other reagent does not guarantee increased transfection efficiency. Nevertheless, the TransIT-TKO siRNA transfection reagent is fairly priced, and if used according to the provided transfection protocol, 1 mL should be good for approximately 500 transfections in 24-well plates. Overall, the TransIT-TKO siRNA transfection reagent works nicely, and the transfection protocol is easy to follow. Those who wish to work with RNAi should therefore consider using this product for transfections of dsRNA oligonucleotides.
Joe F. Lau
Graduate Student
Mount Sinai School of Medicine, NY.