RNA interference (RNAi) uses short strands of synthetic ribonucleic acid (RNA) to silence or “knock down” genes implicated in certain phenotypes—most commonly (but not limited to) diseases. The “interference” occurs when interfering strands bind to complementary, naturally occurring RNA according to standard base-pairing rules. Unlike antisense technology, which operates on DNA, RNAi works by silencing RNA, which is the immediate precursor of proteins implicated in the phenotype of interest.
Reagents consist principally of the interfering RNA construct and a transfection agent for introducing the RNA into cells. The most commonly used interfering RNAs are the short (19 to about 25 nucleotides) interfering RNAs (siRNAs) and short hairpin RNA (shRNA). Approximately 75 percent of the RNAi reagent market uses siRNA, according to Chris Cunning, Ph.D., senior manager of market development at Invitrogen (Carlsbad, CA). Both reagent types bind to complimentary sequences on genes.
shRNA reagents are introduced in plasmid format, which means the target cells can incorporate the silencing agent into their genome and pass it on to offspring. “shRNA is absolutely needed when the phenotype takes longer than about two weeks to develop,” says Steven Suchyta of Sigma-Aldrich (St. Louis, MO). shRNA may also be introduced into cells via a virus transfection agent. Some vendors offer lentivirus transfection reagents that provide long-term, stable knockdown in almost any mammalian cell. Vendors also offer peptide-based reagents.
Reagent companies sell RNAi reagents as “virtual kits” consisting of the shRNA or siRNA sequences and an appropriate transfection reagent. Vendors usually guarantee that a certain percentage of multiple knockdown constructs they sell for a particular target RNA will succeed. Users can monitor the progress of their knockdown by performing a before-and-after Western blot to determine if the protein coded by the putative knockdown gene is still being produced.
Length is a critical attribute of siRNA and shRNA reagents. In nature, interfering RNA species are usually between 20 and 25 nucleotides in length. Longer constructs could theoretically work better since they cover a greater fraction of the target gene, but 20 to 25 nucleotide lengths are ideal for entry into RISC complexes. Furthermore, larger genes tend to be recognized by cells as viruses, which induces an undesirable interferon response.
RNAi may eventually have greater impact on biology than polymerase chain reaction (PCR), although RNAi can be considered more complex than PCR.
The main obstacle is introducing the interfering RNA sequence into the cell, into the location of the target gene, and then getting it to bind to and inactivate the target. Success with one sequence, transfection agent, and cell does not guarantee success when one variable changes.
Delivering interfering RNA into whole organisms (vs. cells) presents even greater challenges, but the potential rewards are also high. Whole-organism or whole-tissue knockdowns would provide new opportunities in drug testing and, eventually, for human therapy.
ExpressArt® mRnA Amplification Kits
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DsiRNA Gene Silencing
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Integrated DNA Technologies
nCounter® Mouse miRNA expression assay Kit
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Thermo Fisher Scientific