RNAi screens for functional genomics typically look for loss- or gain-of-function phenotypes. They currently have many applications, including target discovery and validation, lead identification and optimization, mechanism of action discovery, predictive toxicology, and biomarker identification.
While reagents are gaining ground, technical difficulties remain in how best to perform and analyze these assays.
“RNAi reagents are being used ubiquitously,” says David Root, Ph.D., director of the RNAi Platform and project leader of the RNAi Consortium (TRC) at the Broad Institute. Dr. Root was among a handful of scientists and corporate partners who created one of the early genome-wide libraries of shRNA constructs (called TRC1). As of 2011, TRC2 has expanded the library (to 300,000 shRNAs) and has measured the knockdown performance of 100,000 of those constructs.
To perform RNAi screens, researchers can choose among a growing variety of reagents, including siRNA, shRNA, miRNA, and lncRNA constructs, as well as pooled libraries and constructs coupled to inducers. Synthetic siRNA has a transient effect, and it is eventually degraded. shRNA can be introduced into a cell within a lentiviral backbone, which allows the construct to be incorporated into the host cell’s genome and then stably expressed. This means the gene is always expressing the shRNA.
“Our philosophy has always been ‘siRNA if you can, shRNA if you must,’” says Christophe Echeverri, Ph.D., CEO and CSO of Cenix BioScience USA. “If the chosen cell system allows for good target knockdown by siRNA transfection, and the timeline needed for the experiments and assays is compatible with the duration of the siRNA effects, then it’ll almost certainly be an siRNA-based study.”
Louise Baskin, senior product manager at Thermo Fisher Scientific, reports that siRNA is one of Thermo’s most popular RNAi reagents, especially in the SMARTpool format. This reagent combines four different siRNAs to the same gene. “SMARTpool reagents are favored by a lot of screeners because it gives them fewer wells per gene—fewer plates to manage, less transfection agent to use.”
shRNA screens have their specific advantages, however. “With always-on gene expression,” Baskin says, “one advantage is that researchers can create stable cell lines and perform, for instance, two-week-long assays.”
RNAi screens can be targeted or genome-wide. Thermo Fisher sells a variety of constructs, from gene family subsets to entire human or mouse genomes. Kinases and ubiquitin ligases are popular, Baskin says. “People can work either based on gene families, gene pathways, areas of interest like DNA damage response,” or they can perform “larger, unbiased investigations” that interrogate the whole genome.