RNAi methods and screening: RNAi based high-throughput genetic interaction screening

Abstract

Expanding on decades of mutational analyses, numerous genome-scale RNAi screens have now been performed in C. elegans, leading to estimates that the majority of genes with essential functions that can be revealed by single-gene perturbations have already been identified in this organism. To build on this basic foundation and uncover condition-dependent or combinatorial effects of non-essential genes will require even higher-scale screening. Here we describe a method for performing high-throughput RNAi-based screens in C. elegans in liquid in 96-well plates, and we explain how to systematically test for enhancement and suppression of temperature-sensitive mutations. This chapter covers our entire set of protocols, from setting up the experiment and screening schedule, to scoring the results. The rapid acquisition of high-quality images of each experiment allows the management of a large number of samples per screening cycle and opens up new possibilities for quantitative scoring, computerized image analysis, and the ability to review results independent of the time constraints that are associated with large-scale screening.