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.
RNA interference (RNAi) uses short double-stranded RNAs (dsRNAs) to selectively knock down gene expression. Short interfering RNA (siRNA), delivered exogenously or expressed in transfected cells, is taken up by the multiprotein RNA-induced silencing complex (RISC) in the cytoplasm, guiding that complex to identify and cleave the cognate mRNA, marking it for degradation.
When it was discovered, RNAi was a game-changer, obviating the need for cumbersome knockout mouse strategies. Today, other options for quick knockouts now exist—most notably CRISPR/Cas and TALENs. Yet RNAi remains a popular choice for tweaking gene expression, and researchers have devised several strategies to manipulate and exploit the system for both research and therapeutic applications. Here we examine their pros and cons.
Oligo-archy
The most common approach for RNAi is complexing siRNA with a lipid- or polymer-based transfection reagent and then delivering that to the cell, says Louise Carr Baskin, senior product manager for Dharmacon, which is now part of GE Healthcare Life Sciences. “Generally speaking, siRNA comes ready to use.”
The technology has been around for more than 10 years. In most cases, users need simply specify the gene of interest, and the vendor supplies the siRNA sequence. "Most of them are wet lab-validated to show they knocked out that particular gene, at least on an expression level,” says Vikram Devgan, global head of biological research content marketing at Qiagen, which includes siRNAs in its RNAi portfolio.
Because each siRNA targets a specific region of a given transcript, most customers order and test several designs (one to four usually) for a given gene to determine which gives the best results in their hands, Devgan notes.
Then, once researchers have settled on a particular siRNA sequence, “the second step is making it better,” says Ali Javed, director of R&D for Gene Link. By substituting modified bases for the traditional A, U, G and C, and phosphorothioate linkages for traditional phosphodiester linkages, for example, duplex specificity and resistance to nuclease degradation can be increased. There are even modifications—like the addition of cholesterol or conjugation to a peptide—that can increase the ability of the siRNA to enter the cell in the first place. Many vendors offer altered oligos; some, like Gene Link, let researchers choose from a long list of modifications, while some “other companies keep it proprietary,” notes Javed.
Pool party
In many cases, a single siRNA is sufficient to knock down a gene, says Javed. Nonetheless, whether it’s to find the right siRNA, to hedge bets that it’s not some off-target effect generating a phenotype or to do large screens, many researchers use pools of multiple siRNAs to generate their data points, notes Baskin. Dharmacon, for instance, pools four siRNAs to the same target in so-called “SMARTpool” reagents for both mouse- and human-genomic targets. “Do a screen of 18,000 genes, get 100 really interesting hits with your SMARTpool, then take those 100 and do the four separate experiments to look at each of the four component siRNAs individually,” Baskin says.
The cognate mRNA template itself also can serve as the source from which to generate a pool of siRNAs. Sigma-Aldrich’s MISSION® esiRNAs (endoribonuclease-prepared siRNAs) are prepared by digesting a PCR product of the target-gene transcript. “Now you have 15 [to] 30 siRNAs in small volumes, small molarity, all against that endogenous target, the idea being that you minimize the possibility of off-targeting while at the same time [getting] 30 independent shots at this transcript,” says Shawn Shafer, functional genomics market segment manager at Sigma-Aldrich.
Life Technologies’ BLOCK-iT™ Complete Dicer RNAi Kit enables labs to generate esiRNA-like siRNA pools (termed “d-siRNA”), as well.
According to Shafer, esiRNAs reflect the actual transcript in the cell being investigated rather than the current (and possibly incorrect) annotation in a genome database. “If a couple of bases are changed in the wrong spot, you’re not going to hit that transcript.”
Renewable resource, continuous expression
Consumables like siRNAs are fine for one-shot screens to see if knocking down a particular gene yields a phenotype in your system. But to do more complementary or downstream experiments—to query that gene’s role in a pathway, for example—it’s nice to have a renewable resource.
A group in Boston, led by Judy Lieberman at Harvard University, recently produced a pool of siRNAs—which the group calls pro-siRNA—in E. coli-transfected with the gene for a viral siRNA-binding protein and a long-hairpin dsRNA. Using this pool, they knocked down target-gene expression by about 90% in HeLa- and HCT116-derived human cell lines [1]. An accompanying commentary remarked that because both pro-siRNA and esiRNA are generated by RNAse III activity, they should have similar coverage, yet “pro-siRNAs are easier to make,” and “the pro-siRNA source is renewable” [2].
Short-hairpin RNAs (shRNAs) are renewable in two senses of the term.
shRNAs are processed into siRNAs by the endogenous RNAi machinery. Plasmids or lentiviral particles coding for such shRNAs—and often selectable markers, as well—can be introduced into cells by standard techniques, allowing the cells to either continuously or inducibly manufacture siRNA. Plasmids can be stored as bacterial glycerol stocks; virally transduced cells can be propagated, aliquotted and frozen, so the lab can generate them at will.
Vectors into which the shRNA of interest can be cloned are available from several vendors, as are off-the-shelf vectors that already contain the shRNA of interest. The usual plasmid transfection/viral transduction considerations apply, including ease of use and efficiency, difficult-to-transfect cells, stability, safety precautions and immunogenicity.
If you're gearing up to try RNAi for the first time, the options may seem daunting. Fortunately, the technique is simple and relatively inexpensive. Embrace the options, and go knock down some genes.
References
[1] Huang, L, et al., “Efficient and specific gene knockdown by small interfering RNAs produced in bacteria,” Nat Biotechnol, 31:350–6, 2013. [PubMed ID: 23475073]
[2] Blau, JA, McManus, MT, “Renewable RNAi,” Nat Biotechnol, 31:319–20, 2013. [PubMed ID: 23563428]
Image: Viral silencing suppressor bound to an siRNA. (Proteopedia)