Product Focus: RNAi Reagents

Delivering interfering RNA into whole organisms (vs. cells) presents even greater challenges, but the potential rewards are also high.

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Enablers of the Brave New World of RNA Interference

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.

Reagent types

RNAi occurs in nature in most animals through a group of molecules known as microRNAs. RNAi reagents seek to duplicate this process.

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.

But there are differences. siRNA produces transient transfection through the action of a lipid reagent, which encapsulates the siRNA within a liposome or soap bubble, disrupts the cell membrane, and chaperones the gene into the cell. Transient transfection means the siRNA signal is not carried from one generation to the next. Thus, even if siRNA is introduced at very high transfection efficiency, the silencing gene is eventually extinguished because daughter cells do not carry it. Moreover, only certain cells, such as “immortalized” cancer cell lines, accept siRNA transfection. The advantage of siRNA is that it is relatively facile, and the reagents are easy to work with.

For example, Integrated DNA Technologies (Coralville, IA) keeps about a dozen cationic lipid transfection agents on hand, some of which it sells. Mark Behlke, M.D., Ph.D., chief scientific officer at Integrated DNA, also uses electroporation and peptide reagents, such as Transductin™, which was invented at the University of California, San Diego.

Not all transfections are equal

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. Sigma Aldrich and other companies offer lentivirus transfection reagents that provide long-term, stable knockdown in almost any mammalian cell.

Vendors also offer peptide-based reagents

Viral transfection is “difficult and labor intensive,” says Invitrogen’s Chris Cunning. “It’s harder to predict what sequences will actually work with them.” shRNA-based interference works about half the time, whereas siRNA is about 90 percent predictable.

In the early days of RNAi work, research groups were much more likely to synthesize their own RNA constructs than they are now. Since RNA synthesis is a tedious process, biologists usually farm out this work to reagent manufacturers. Reagent companies use complex, proprietary algorithms to predict sequences that will work. “The considerations are extremely deep,” says Mr. Cunning, “including toxicology and effect on known, natural microRNAs.”

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.

More important than PCR?

RNAi may eventually have greater impact on biology than polymerase chain reaction (PCR). But Behlke warns that RNAi is “far more complex” than PCR. “Gene knockdowns require a lot more optimization and work since they occur in living cells.”

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. Wholeorganism or whole-tissue knockdowns would provide new opportunities in drug testing and, eventually, for human therapy.

RNAi REAGENTS:

For aditional resources on RNAi reagents, including useful articles and a list of manufact urers, visit www.labmanager.com/reagents

If you’re looking to purchase RNAi reatgens, visit LabX.com to browse current listings.

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Categories: Product Focus

Published In

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Career Building

Published: October 1, 2010

Cover Story

Career Building

While technical ability is essential to becoming a successful laboratory manager, it is not sufficient. Many outstanding scientists or engineers have failed as lab managers. It takes more than just technical ability. What is this more that outstanding lab managers have?