by Catherine Shaffer
Short interfering RNAs, or siRNAs, created a sensation in molecular biology in 2001, when it was discovered that they could induce gene silencing. This powerful new method of "knocking out" proteins has become a valuable research tool, as well as a hot property in the field of gene therapy. Which means that there is a great demand for tools and techniques for transfecting siRNAs into cells, both in vitro and in vivo. Major priorities for siRNA transfection are high transfection efficiency and low toxicity. This is true in the case of any transfection, but particularly for siRNA knock-downs where the issues of toxicity and transfection efficiency are intertwined. It can be difficult to distinguish true gene silencing from cell toxicity. "Having a reagent that has low toxicity is even more important when you're talking about RNA interference. You're looking for knockdown of the gene itself. You want to make sure that gene inhibition is due to siRNA or microRNA, not due to toxicity from your reagent," says Mary Kay Bates, technical support manager for Mirus Bio.
Mirus Bio is a biotechnology company focused on developing nonviral gene therapeutics. They developed the first reagent for transfecting siRNA—TransIT®-TKO—to support their own research. Both Mirus’ TransIT®-siQUEST and TransIT®-TKO reagents are engineered specifically for siRNA transfection. The proprietary formulas include a combination of lipids and polymers that maximize transfection efficiency while minimizing cellular toxicity across a wide range of cell types. In fact, they offer a combo product, TransIT®-siPAK, so that customers can sample each reagent to choose the best one for their application. These reagents also have the advantage of using less siRNA and less transfection reagent, which is more economical.
Millipore also offers a transfection reagent designed especially for siRNA. Their siIMPORTER siRNA transfection reagent, in combination with the siRNA/siAb Assay Kit, is guaranteed to deliver siRNA mediated gene knockdown with high transfection efficiency and low cytotoxicity. Scientists from Germany used siIMPORTER to inhibit Dickkopf-1. This knocked out the bone destructive processes in rheumatoid arthritis, and stimulated bone-forming activity as in osteoarthritis, revealing Dickkopf-1 as a master regulator of joint remodeling.1
There are actually two ways that siRNA can be transfected. One is by directly using a transfection reagent tailored to very short stretches of DNA. A second method is by cloning the siRNA into a plasmid construct. "Millipore's plasmid-based siRNA constructs place RNA polymerase III promoter upstream of a DNA sequence that, when transcribed, folds back on itself to form a short-hairpin RNA (shRNA). The shRNA is ultimately processed by the cell to form a functional siRNA," says Antonia Shtereva, associate product manager for Millipore. The Millipore siRNA expression plasmids are validated in cell culture before release. Each plasmid is guaranteed to knock-down its intended target by at least 70%.
Mirus Bio's TransIT®-LT1 Transfection Reagent can be used to transfect plasmids carrying siRNAs, and this method is often preferable if long-term expression of the gene of interest is desired. In addition, researchers are increasingly interested in working with other types of small RNAs other than siRNA, such as microRNA. The TransIT siRNA transfection reagents have proven to be equally effective when used with these other RNAs.
Invitrogen's Lipofectamine RNA Max was designed specifically for the delivery of synthetic siRNA. Says Po-Tsan Ku, PhD, Product Manger for Transfection at Invitrogen: "There was a need for siRNA transfection reagents that can have a high transfection efficiency and achieve high gene silencing with low siRNA concentration, across a broad range of cells ... those are the goals we had in mind when we developed this product." Lipofectamine RNA Max, a cationic lipid based reagent, works by complexing with synthetic siRNA oligos, and delivers them across the plasma membrane into the cell.
One advantage of Lipofectamine RNA Max is that it has a broad useful range of concentration; "For example, if a customer were using it, adding more than they really needed to," says Henry Chiou, PhD, technology area manager in R&D for Transfection, "this reagent allows them to do that without showing a lot of cell death or disruption of biological processes. Other reagents have a narrower range, and the user has to be a little bit more careful that they're using a proper amount of reagent." This is a boon to customers who may be entering the RNA field for the first time. "Even without optimization, you can achieve very good gene silencing results," Chiou adds.
The flip side of the range issue is that it also permits variation in the density of cells in culture. When there are fewer cells in culture, those cells are more vulnerable to toxicity. Having some "wiggle room" in terms of the range of concentration makes it possible to use sparser cell cultures, either to conserve materials, or because of experimental limitations.
References:
1Diarra D. et al., “Dickkopf-1 is a master regulator of joint remodeling,” Nature Medicine 13(2):156-63, Feb 2007.
