CCG siRNA Screening

 

Whole genome siRNA screening now in CCG

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.
 
Why do siRNA screens? 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 or 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 (1).  Lipid transfection, once the optimal reagent and conditions have been identified, 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.
 
The CCG is excited to host this important technology and we thank the contributors who helped us obtain it (2). Please contact the CCG if you think that your studies would benefit from this cutting edge approach.
 
 
References:
(1) Mohr S, Bakal C, Perrimon N. Genomic Screening with RNAi: Results and Challenges (2010) Ann Rev Biochem, 79:37-64.
(2) Supported by The Life Sciences Institute; UMMS-Office of the Dean; U-M Comprehensive Cancer Center; UMHS-Department of Pathology; Michigan Comprehensive Diabetes Center; Michigan Diabetes Research and Training Center; MHS-Department of Internal Medicine; Michigan Center for Translational Pathology; Michigan Institute for Clinical & Health Research.

The HTS Core has Dharmacon’s human and mouse iGenome SMART Pools (pdf) available for screening in 384-well format. The core utilizes RTP (reverse tranfection protocol) and many assay detection methods are available with the HTS core instrumentation. The library is organized by gene subsets and discounts for Dharmacon reagents are available. smswaney [at] umich.edu ( )

Please review the following reference material about RNAi screening: NIH RNAi Assay Guide , Pub Med article

Introduction videos about siRNA screens: 

Overview

RNAi Details

Nature Video