Many scientists once considered high-throughput screening (HTS) as the key to unlock many scientific questions, from unraveling biochemical pathways to finding new drugs. Undoubtedly, HTS plays a role in many experiments today, but some scientists remain dissatisfied with the payoff.
In the February 9, 2007, Nature Reviews Drug Discovery, a scientific duo from Abbott Laboratories, Abbott Park, Ill., wrote that “the return on investment in terms of marketed products has not met expectations.” Much like HTS, the explosion of techniques related to RNA interference (RNAi) also promises many new findings. To realize those results, scientists can now combine HTS and RNAi.
To make this combination productive, scientists need the right tools. In the February 2007 Genomics, X.D. Zhang of Merck Research Laboratories, West Point, Pa., notes, “The application of genome-scale RNAi relies on the development of high-quality RNAi HTS assays.” In 2006, Merck scientists screened 600,000 RNAi wells in search of potential drug targets.
To power such projects, many companies already market HTS-RNAi tools. For example, the Ambion Inc., Austin, Texas, siPORTer-96 Electroporation Chamber can be combined with the Bio-Rad Laboratories, Hercules, Calif., Gene Pulser Xcell generator to “push” short interfering RNA (siRNA) into cells with an electric pulse. According to Ambion’s literature: “You will be able to detect RNAi-induced gene silencing just a few hours after delivery.”
|Developing a Database
As RNAi screening spans genomes, scientists need ways to keep track of all of the information. A team of researchers at the German Cancer Research Center in Heidelberg hopes to solve some of that problem. In the 2007 database issue of Nucleic Acids Research, Thomas Horn and his colleagues reported on their GenomeRNAi. “The database,” write the authors, “integrates the sequence information of the RNAi reagents with phenotypic information based on genome-scale and genome-wide published RNAi screens. It contains 90,998 RNAi probes that have been designed by various groups.” Specifically, these probes came from phenotypes generated in RNAi screens of cultured Drosophila cells.
Anyone interested can access GenomeRNAi online (www.dkfz.de/signaling2/rnai/ernai.html). Moreover, this tool offers a range of ways to query the database. Users can search by using a gene name, an RNAi probe ID, or a phenotype. In addition, the developers provided advance query capabilities, such as sequence-homology searches. Users can also perform sequence-similarity searches. A sequence of Drosophila nucleotides can be entered, for example, and the database finds the RNAi probes that overlap that sequence. As the authors conclude: “The GenomeRNAi database presents phenotype information of large-scale screens in the context of associated sequence information.” Eventually, GenomeRNAi will also include phenotypes from humans and other species.
Moreover, many assays focus on families of targets. The Qiagen Inc., Hilden, Germany, Human GPCR siRNA Set, for instance, targets 471 G-protein coupled receptors. Sometimes the focus aims at specific species. At Invitrogen Corp., Carlsbad, Calif., for example, the Stealth RNAi Collection aims at human and mouse targets. To simplify HTS, these collections comes in 96-well plates.
Getting accurate results from an HTS-RNAi combo, however, also depends on the analysis. At the 14th annual Molecular Medicine Triconference in San Francisco, Dmitry Samarsky of Dharmacon Inc., Lafayette, Colo., discussed statistical approaches to RNAi-based screens.
In an abstract to his presentation, he wrote: “Given the size of the data sets associated with genome wide screening, procedures that assist researchers in data analysis and assay selection are crucial.” In fact, being able to share results across experiments requires the development of standards for HTS-RNAi screening, and Dharmacon is leading such an effort.
Dharmacon put together a group of researchers—based on people who had bought one of the company’s genome-wide siRNA libraries, such as the siGENOME Collection—to create guidelines. These will be called the Minimum Information about an RNAi Experiment, or MIARE, and more information can be found at the group’s website (www.miare.org).
Despite the growing collection of HTS-RNAi assays, scientists cannot expect miracles. In a survey of ten laboratories using the same reagents and protocols, Dharmacon found considerable variability in the results. In the future, standard statistical methods must be incorporated in screening to accurately compare results.
About the Author
Mike May, PhD is a publishing consultant for science and technology based in Minnesota.
This article was published in Drug Discovery & Development magazine: Vol. 10, No. 3, March, 2007, pp. 16-17.
Filed Under: Drug Discovery