siRNA Screening

The CCG is recognized for its chemical screening resources, but we now have available a whole-genome mammalian genetics screen capability. In 2009, we acquired the Dharmacon siGenome human and mouse whole genome smart pool libraries. They include pools of 4 oligonucleotide small-interfering RNAs (siRNA) against each of ~18,000 human or ~17,000 mouse genes. They can be used to do an unbiased analysis of genes involved in complex biological processes.

You may know the basic elements of a cellular signaling pathway but would like to know what other processes are required for or modulate that signaling process. Alternatively, in different cell contexts (e.g. pancreatic beta cells vs. cardiac myocytes) the process may utilize different subtypes of key signaling elements. A whole genome siRNA screen would point you to both unknown genes affecting the process and to specific subtypes of large gene families that are critical in your cell system. This is of importance biologically, but it can also reveal novel drug targets that may be attacked chemically in a drug discovery effort. To support latter, the CCG also provides a “subset” screening library termed the “druggable genome” collection, which includes many common pharma drug targets such as GPCRs, kinases, phosphatases and ion channels. It also includes proteases and elements of the ubiquitin proteasome mechanisms.

 

The CCG siRNA collection uses the oligonucleotide RNAi approach that has been applied to a large number of cell systems and biological problems. Once the optimal reagent and conditions have been identified, lipid transfection is quite efficient and the inclusion of 4 separate siRNA against each gene generally provides good knock-down. With the purchase of the Dharmacon siGenome, the CCG has become a member of the Global siRNA Initiative, with access to a network of other researchers and technical tips that can facilitate study design.

 

The key first step in a siRNA screen is to identify a screening readout that can be readily tested in 10,000+ samples. We generally recommend that siRNA studies be done in triplicate to avoid false positives and false negatives in light of the requirement for lipid transfection. Any cellular readout that can be detected in the CCG (or potentially in your lab if you have a plate reader system or an efficient scoring method) can be used for siRNA screens. Luciferase reporters, fluorescence or FRET measures, second messenger assays, or secretion (e.g. Alpha-LISA) are all amenable to HTS. Cell morphology studies using the CCG’s high content system (ImageXpress Micro) are also feasible. Contacting the CCG early may help you design the best approach for our existing capabilities and will let us assist you on optimizing your method.