by Jeffrey M. Perkel
Small regulatory RNAs, such as microRNAs and the short-interfering RNAs (siRNAs) that drive RNA interference, are not only interesting research subjects in their own right; they are invaluable research tools, as well. Researchers use these tiny nucleic acids to degrade or at least dampen expression of complementary mRNAs in vitro and in vivo without the need for extensive genetic manipulations.
Small wonder, then, that four years since Andrew Fire and Craig Mello won the Nobel Prize for their discovery of RNA interference, research in the field continues at an astonishing clip. According to the ISI Web of Knowledge, some 9,044 articles were published in 2009 on either "RNA interference" or microRNAs, up from 6,828 the year before.
Of course, to reap the benefits of short-interfering RNAs and microRNAs, scientists need a way to get these molecules into cells in the first place, and therein, as they say, lies the rub. Researchers have had tools to ferry kilobase-sized DNA plasmids across cell membranes for decades. But those tools don't work quite so well when it comes to 20-odd-nucleotide RNAs.
According to Louise Baskin, product manager for the Thermo Scientific Dharmacon siRNA and transfection product lines, it's like the difference between delivering a pizza on a scooter as opposed to an 18-wheeler truck; one is simply a more appropriate transit for the payload.
"For a long time the only thing people had available was a fleet of semitrailers, which were optimized for a very different cargo," she explains. "The advent of RNAi as a research tool demanded reagents that are optimized for small RNA transfection, which are more efficient and gentle to the cells."
These days scientists can more or less take their pick of suitable delivery vehicles, and that's a good thing, because otherwise, unintended consequences can crop up. A surfeit of siRNA can lead to off-target effects, for instance, while inappropriate transfection reagents can lead to reduced delivery efficiency, increased cytotoxicity, and inflammatory responses.
There basically are three approaches to delivering small RNAs (or really, any nucleic acids) to cells: transfection reagents and chemical agents, electroporation, and viruses. Most researchers, for reasons of simplicity and cytotoxicity, use transfection reagents for small RNA work—between 60% and 80%, according to Constanze Kindler, senior global product manager at Qiagen. That's because transfection reagents—generally, lipid concoctions that form complexes with the small RNAs and which are taken up by endocytosis—are simple to use, relatively non-cytotoxic, and amenable to high-throughput screening and automation.
These reagents work best on rapidly dividing, adherent cells—cell lines, basically. But many of the most interesting cells from a researchers' perspective—slow-growing cells, stem cells, suspension cells, and primary cells—don't respond well to lipid-based reagents, at best refusing to take up the complexes, at worst, developing adverse reactions. In such cases, viruses or electroporation may be the only option.
"I think the biggest unmet need right now is a really easy-to-use lipid-like reagent for suspension or primary cells," says Kristin Wiederholt, senior research and development manager at Life Technologies. "Electroporation and the different viral approaches will work, but … it's a lot more complicated. Lipid protocols are much more straightforward." And, she adds, neither electroporation nor viral approaches are easily scaled.
Virus work can be especially complicated. Available commercially either as empty, insert-your-sequence-here vectors (e.g., the BLOCK-iT™ Lentiviral RNAi Expression System from Life Technologies), shRNA expression vectors (e.g., Thermo Scientific GIPZ shRNA constructs), or pre-packaged virions (e.g., Thermo Scientific SMARTvector 2.0 Lentiviral shRNA particles), viral approaches require the engineering of constructs that will express the small RNA as either a precursor miRNA or short-hairpin RNA (shRNA), which are then expressed in the cell and processed to form a functional siRNA or microRNA mimic. Between the labor required to produce the infectious viruses and the transduction itself, the process can take weeks and is not easily automated.
Electroporation approaches are simpler, but still less so than lipid protocols, says Wiederholt. They also are relatively cytotoxic—the electrical pulses that induce pore formation in cellular membranes can kill a high percentage of the cells if not tuned just so—and often require a considerable number of cells per experiment.
To circumvent those problems, Life Technologies' Neon system uses a specialized consumable pipette tip instead of the standard electroporation cuvette. This tip, says Wiederholt, "simplifies the workflow": Simply pull up your cells and nucleic acid using the Neon Pipette (as few as 10,000 cells per experiment), insert the tip end-down in the electroporator, press start, and replate. Thanks to its design—for instance, the surface area of the electrode is smaller than a standard cuvette—the conditions are gentler than conventional electroporation, resulting in higher viability, says Wiederholt.
Most researchers, though, opt to use cationic or amphipathic lipid transfection reagents, and several are available, including Life Technologies' Lipofectamine™ RNAiMAX, Qiagen's HiPerFect, Thermo Scientific DharmaFECT®, Roche Applied Sciences' X-tremeGENE siRNA transfection reagent, and Polyplus Transfection's INTERFERin™ and screening-ready INTERFERin-HTS.
The protocols are simple, cookbook chemistry: mix nucleic acid with reagent, wait a few minutes, apply to cells, and assay a few days later. But be prepared to optimize, says Baskin; the goal is to deliver just enough siRNA, with just enough reagent, to silence the gene of interest without collateral molecular damage—but transfection efficiency will vary by cell type, confluence, siRNA concentration, and plate format, among other factors.
On its website, for instance, Polyplus demonstrates that silencing efficiency can vary widely over a range of siRNA concentrations, from 50% inhibition at 10pM siRNA, to 90% at 1nM siRNA. Life Technologies presents data on its website showing relatively constant knockdown of p53 using 1nM siRNA and a 10-fold range of Lipofectamine RNAiMAX with only a 10% to 20% reduction in viability. Alternatively, researchers can use any of Thermo's four DharmaFECT flavors to better their chances of finding a reagent that works best in their hands; "a one-size-fits-all approach" is less likely to accommodate the biology of all different cell types, Baskin explains.
Not all transfection protocols require a dedicated delivery tool. Thermo Scientific's Accell® siRNA portfolio is a pre-designed collection of siRNAs (covering all known human, mouse, and rat genes) that are chemically modified for transfection in the absence of exogenous reagents.
Intended for cells that are especially resistant to transfection—primary neural, immune, and cardiac cells, for example—Accell siRNAs are adorned with proprietary modifications that both stabilize the molecules (to protect against nuclease digestion, for instance) and facilitate transmembrane delivery, says Baskin.
For those researchers specifically interested in miRNAs, the delivery options generally are the same (though Polyplus recommends miRNA researchers use its more versatile jetPRIME™ formulation rather than the siRNA-ready INTERFERin). As with siRNAs that are delivered with a lipid-transfection reagent, libraries of miRNA mimics and inhibitors are available from the likes of Thermo Scientific (miRIDIAN mimics and hairpin inhibitors) and Qiagen (miScript mimics and miScript miRNA inhibitors). For viral delivery, Thermo Scientific offers its miRIDIAN shMIMIC lentiviral microRNAs.
If there's one key unmet need remaining in the small RNA marketplace, says Baskin, it is reliable in vivo delivery. That, she says, is "the silver bullet that every small-RNA therapeutic group is seeking." And, despite reported successes with in vivo RNAi in the research space, even animal models can be refractory to exogenous RNA, she notes. "When you talk about animals, you have potentially hundreds of different cell types, and lots of environmental variables that cell culture does not."
That said, Polyplus does offer tools for non-viral in vivo siRNA delivery. The company's in vivo jetPEI™ formulation, for instance, "is compatible with most delivery routes for siRNA administration," says Erbacher, while its jetSI™ reagent specifically enables delivery to the brain.
Susan Magdaleno, another senior research and development manager at Life Technologies, says that when it comes to small RNA delivery, the key is to consider your downstream applications. "You do have to put a lot of thought into choosing a delivery system to match to your biological goals," she says. "What are you trying to get done?"