Easing siRNA Transfection

Image: Courtesy of Ambion SILENCE! HeLa cells transfected with Cy3-labeled GAPDH or Scrambled siRNAs using Ambion's siPORT Lipid Transfection Agent were harvested 48 hours post-transfection. Red: Cy3-labeled siRNA; blue: DAPI-stained nuclei; green: GAPDH protein. Introduction of the scrambled control siRNA (left) had no effect on GAPDH protein expression, whereas transfection of an siRNA targeting GAPDH (right) resulted in drastic reduction in GAPDH protein levels. Much attention has been paid recently to RNA interference (RNAi), a technique in which exogenous, double-stranded RNAs (dsRNAs) are introduced into a cell to specifically destroy a particular mRNA, thereby diminishing or abolishing gene expression.1 The technique has proven effective in Drosophila, Caenorhabditis elegans, plants, and recently, in mammalian cell cultures.2 To make the technique work in cultured mammalian cells, researchers must deliver small interfering RNAs (siRNAs), which are dsRNAs of 21-25 nucleotides, to the cell. They can use a wide range of standard transfection reagents optimized for DNA and RNA, but according to Kathy Latham, RNAi product manager for Austin, Texas-based Ambion, reagents designed to deliver mRNAs, for instance, are not adept at transfecting smaller siRNAs. "It's like the difference between plasmids and oligos," she says, referring to manufacturers' offering different formulations to transfect plasmid DNA and shorter oligonucleotides. As a result, both Ambion and Madison, Wis.-based Novagen have recently unveiled reagents specifically designed to deliver siRNAs to cells. Ambion offers two products: siPORT Amine, a polyamine-based reagent, and siPORT Lipid, a mixture of neutral and cationic lipids. Investigators may purchase these compounds separately or as part of the Silencer™ siRNA Transfection Kit, which contains both reagents as well as positive and negative siRNA controls (targeting GAPDH mRNA). According to Latham, Ambion's dual formulations reflect the fact that different cell lines exhibit different transfection efficiencies; some will perform better with the amine reagent, whereas others will prefer the lipid formulation. Data on Ambion's Web site appears to support this assertion. COS-7, CHO-K1, 293, and BJ cells are best transfected with the amine reagent, whereas HeLa-S3 and MCF-7 prefer the lipid formulation. NIH-3T3, HT-29, and A549 cells perform well with either reagent. Novagen has adopted a different approach, combining both amine- and lipid-based reagents in a single formulation called RiboJuice™. The mixture of these two classes of compounds in one mixture evidently makes RiboJuice effective on a broader range of cell lines than either of Ambion's reagents alone. Novagen has successfully tested its RiboJuice reagent with Neuro-2A, BHK, COS-7, MCF-7, NIH-3T3, A549, HeLa, HEK293, CHO, HepG2, and L6 cells. Both companies claim that these reagents will work on adherent as well as suspension cells. Using these reagents is relatively simple. The researcher dilutes the transfection reagent in a serum-free medium, such as GIBCO BRL's Opti-MEM®, adds the siRNA, and then, following a short incubation period, overlays the mixture onto the cells in a dish. The cells are then incubated with the transfection complexes for up to 72 hours, at which time they are harvested and tested. Optimization is a critical part of RNAi research. According to Latham, Ambion recommends scientists first optimize the cell-plating density, then the amount of siPORT reagent to use, and finally the quantity of siRNA. Another factor to consider is the sequence of the siRNA itself. In a Novagen technical note,3 the company reports that five siRNAs were synthesized to target different regions of ß-galactosidase. While three of the five efficiently downregulated gene expression, two had no effect whatsoever. "The results underscore the importance of testing more than one siRNA for any given target mRNA," the report concludes. A final consideration is whether to have serum in the growth medium. Both Ambion and Novagen stress that the transfection complexes should be formed in the absence of serum, but researchers can add siPORT Amine and RiboJuice to cells in serum-containing medium. Users of siPORT Lipid, on the other hand, should incubate the cells and transfection complexes in serum-free medium for four hours before applying normal growth medium. --Jeffrey M. Perkel References 1. A. Fire et al., "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans," Nature, 391:806-11, 1998. 2. S.M. Elbashir et al., "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells," Nature, 411:494-8, 2001. 3. S. Hayes, F. Bruggink, "Targeted suppression of gene expression using siRNA and RiboJuice siRNA transfection reagent," inNovations, 14:9-11, 2002.

Easing siRNA Transfection

Interested in reading more?

Become a Member of

Image: Courtesy of Ambion
SILENCE! HeLa cells transfected with Cy3-labeled GAPDH or Scrambled siRNAs using Ambion's siPORT Lipid Transfection Agent were harvested 48 hours post-transfection. Red: Cy3-labeled siRNA; blue: DAPI-stained nuclei; green: GAPDH protein. Introduction of the scrambled control siRNA (left) had no effect on GAPDH protein expression, whereas transfection of an siRNA targeting GAPDH (right) resulted in drastic reduction in GAPDH protein levels.