Meteorologists would be hard-pressed to produce a complete list of hurricanes that the ocean currents might whip up next season. Fortunately, making predictions of which diseases could come storming into our lives is fast becoming a reality. The efficiency of RNA inhibition (RNAi) has allowed researchers to study gene expression at a quicker pace. With a new generation of RNAi products designed for high-throughput, you could very possibly prevent nature’s tempests from blowing away your health.
This exciting prospect wouldn’t be as realistic at the lethargic pace of gene knockouts. Taking from months to years, this approach is not only slow, but also requires the expense of labor and dealing with animal models. In addition, gene knockouts eliminate “gene expression throughout the entire organism, and cannot be used for studying developmental or cell type–specific effects unless tissue-specific knock-outs are employed,” according to a paper published in the April 12, 2005 issue of the journal Blood. “In theory, these problems could be resolved by adapting RNAi techniques to silence gene expression [for short-term studies].”
Life scientists first noticed such gene-silencing effects in 1928, when the upper leaves of tobacco plants were resistant to viral infection. Decades of study have revealed that viral infection leads to the production of double-stranded RNA (dsRNA) intermediates, which can silence the expression of the complementary genes of the virus. “This defense against foreign genetic material is one of several physiologic pathways that are induced by naturally occurring dsRNAs in a wide variety of eukaryotic organisms including fungi, plants, and animals,” according to the Blood paper.
Today, researchers synthetically produce dsRNA strands that contain the sequence of the target gene. Once transfected into cells, dsRNA encounters the enzyme Dicer, which chops up the dsRNA into 20- to 25-nucleotide strands called small interfering RNAs (siRNA). The siRNAs aggregate into endoribonuclease-containing complexes called RNA-induced silencing complexes (RISCs). After ATP-generated unwinding, the siRNAs guide the RISCs to the complementary mRNA molecules and destroy them, preventing translation.
To assist in gene-silencing experiments, companies have developed a wide range of tools, reagents, and kits. The various plasmid and virus vectors that carry the DNA code, which the cell’s transcription machinery can use to produce the desired siRNA, are very popular. Employing vectors ensures the type of long-term expression that you require for high-throughput, automated lab settings. You can also purchase the convenience of a number of gene expression cassettes—for both linear and circular vectors—with specifically designed promoters and other features geared toward successful RNAi experiments.
If you prefer to directly transfect dsRNA or siRNA into cells, you can choose from the wide selection of libraries that feature entire genomes, to those that focus on specific tissues or proteins. The market also offers reagents that are pre-aliquoted into 96-well plates, along with software and other tools to get your large-scale screening off the ground. Some of these systems have even been designed with the power to infiltrate primary and difficult-to-transfect cell lines, as well as the flexibility to transfect all types of nucleic acids, even microRNA.
Try to spot these products in the list below. They will certainly calm the seas of your RNAi frustration, and get you moving as fast as any gale-force winds.
For screening the human genome from the experts in high-throughput RNAi. The Human Whole Genome siRNA Set V1.0 offers: High knockdown and minimal off-target effects using the HiPerformance siRNA Design Algorithm, faster discovery using off-the-shelf siRNAs for all human genes, flexible formats including modules, pools, and different scales.
Based on the well-established Nucleofector technology amaxa has developed a system for the highly efficient transfection of primary cells and difficult-to-transfect cell lines in 96-well format. The Nucleofector 96-well Shuttle System consists of three components, the 96-well Shuttle, the Nucleofector II Device and a laptop. Driven by the Nucleofector II, the 96-well Shuttle can deliver DNA directly into the nucleus, allowing even non-dividing primary cells (such as resting T cells or neurons) to be efficiently transfected. siRNA delivery into the cytoplasm, on the other hand, also occurs with up to 99% efficiency. Optimal nucleofection conditions depend upon the individual cell type, not on the substrate being transfected. This means that identical conditions are used for the nucleofection of DNA, siRNA or shRNA vectors. So it is a simple matter to switch back and forth between substrates or to perform co-transfections with DNA and RNA together.
