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.
Complete Dicer RNAi Kit
BLOCK-iT™
• Provides an easy alternative to using synthetic short RNA oligonucleotides for RNAi experiments
• Includes an siRNA purification module specifically developed to isolate pure siRNA from the Dicer reaction
• Provides an effective way to screen as many as five genes in a mammalian model
Invitrogen
www.invitrogen.com
Lipid Reagent for RNAi
siLentFect™
• Consists of a proprietary cationic compound and a co-lipid
• Low volumes of lipid required per transfection help to minimize cell stress
• Offers excellent performance at culture densities between 50 and 90% and in the presence or absence of serum-containing medium
Bio-Rad
www.bio-rad.com
Peptide Delivery System for dsRNA
Transductin™
• Uses the cell’s own mechanism for uptake to avoid toxicity and immune response often observed with other reagents
• Works through a mechanism distinct from cationic lipids
• Can be used to deliver dsRNAs to most cell lines, including primary cells and ES cells
Integrated DNA Technologies
www.idtdna.com
siRNA Generation Kit
Turbo Dicer
• Enables easy generation of a large number of siRNAs from full-length target genes
• Allows the user to quickly produce multiple siRNA species against target mRNA
• Contains everything required for preparing double stranded RNA from target gene(s), dsRNA cleavage, siRNA cleanup and transfection
AMSBIO
www.amsbio.com
