Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Whether for large-scale screens or one-off experiments, RNA interference has become an essential tool for probing gene function. In its most basic version, small RNAs (about 22 nucleotide) called small, interfering RNAs (siRNAs) are used to “knock down” a gene by interacting with complementary mRNA, ultimately targeting the messenger for destruction before it can be translated.
There are myriad ways to introduce siRNA into the cell, from encoding it in plasmids or viruses (generally as a longer RNA that is then processed by the cell) to electrical, magnetic, ballistic or chemical prompts to induce or force its entry. Among these, the method requiring perhaps the least amount of specialized equipment and expertise is transfection. Here, the siRNA typically is complexed with a molecule that neutralizes its negative charge and facilitates its interaction with the negatively charged cell membrane.
siRNA-specific reagents?
Long before the discovery of siRNA, nucleic acids were being transfected into cells using carriers such as CaPO4, polysaccharides like DEAE-dextran, various lipid formulations, peptides and combinations thereof. Today, there are a host of commercially-available reagents marketed exclusively for siRNA transfection or that list siRNA as one of their supported applications. But vendors generally won’t tell you about their product’s composition beyond a description like “cationic lipid formulation,” “polyamine-lipid formulation,” “polymer-based,” “lipid-coated iron oxide nanoparticles” or “blend of lipids and other components.”
Some researchers recommend such si-RNA-specific formulations as the first reagent to try. Claudia Kowolik, former manager of the City of Hope RNAi core in Duarte, California, for example, has run comparisons for a few of the core’s clients. “I would get way better results with [Life Technologies’] Lipofectamine RNAiMAX than the ones that are more for plasmid transfections, like Lipofectamine 2000,” she recalls.
Others, like Chi Yun, director of the NYU RNAi core, say that although she hasn’t performed a systematic comparison, “I haven’t seen a huge advantage in using a transfection reagent just designed for siRNAs.”
For co-transfection of siRNA with a plasmid, though, it’s generally best to use a more generic reagent.
In any case, there’s no substitute for empirical data.
Search, ask and test
So of all the reagents—siRNA-specific or generic, lipid-based or not, a household name or a hot new product, premium or econo-priced—what should you use? It depends in part on the cell type and the species, the downstream assay and whether it’s a co-transfection, how much legwork you’re willing to put in and a few other considerations.
Primary suspension murine cells are notoriously more difficult to transfect into than are adherent cultured human cells, for example, with neurons and macrophages having the reputation of being untransfectable by standard means. Every cell line is different, and conditions that work for one may not work for another.
But there’s a good chance that someone has done something similar. Search the literature. Talk with fellow researchers. Check with vendors—most maintain a list, either online or accessible through tech support, of cell types that have been successfully transfected. (The information may not be available specifically for siRNA, “but it’s pretty useful just to know what cell type they’ve tried and what they consider successful,” says Yun.) RNAi core facilities, too, are great resources for learning which researchers have worked with which cells.
Then get some. Call the vendor and ask for samples or borrow from someone in the building. Test them out on a plate. Within a few days, and with some luck, you’ll know if anything looks promising.
What to look for
There are two main things to watch for when testing a transfection reagent: Does it allow the siRNA to get into the cell? And is it relatively free of negative effects? If the answer to either of these questions is “no,” try another.
The most straightforward way to find out whether the reagent facilitates entry is to perform a positive control experiment: Use it to transfect in an siRNA with a known effect and test for that effect. Many vendors also sell fluorescently labeled, nonspecific siRNA to use as a positive control. Yet some researchers like Yun have had “terrible luck with those”; in her hands, they stuck to the outside of the cells (both with and without reagent) and lit up the entire cell.
To check for off-target effects, “if you want to really make sure everything is fine, you’d have to run a microarray, just to make sure you don’t affect too many other genes,” explained Kowolik. “But that’s going way out of the way, so usually I just looked for healthy cells.”
Of course, after a reagent is chosen it’s best to take time to optimize parameters such as quantities of reagent (use the least that gets the job done) and siRNA, timing of the transfection and how long to wait before testing for effect. This is best done by optimizing around a combination of RT-PCR and a downstream assay, says Yun.
Final considerations
Finally, check with the vendor to make sure the reagents you choose are compatible with your assays and workflow. Some protocols require serum-free medium, for example, or changes of media. And although these types of incongruities may not be insurmountable, if two reagents are otherwise equal, you’ll know which one to choose.