RNAi screens for genes involved in Golgi glycosylation

Abstract

RNAi screening has gained popularity in recent years, due to its usefulness in systematic investigations of biological pathways. Combined with high-content screening and advances in imaging and analysis methods, it can enable detailed genetic characterization of cellular processes such as protein glycosylation, a major function of the Golgi apparatus. Glycosylation concerns about one third of all human proteins and regulates various cellular behaviors. Yet the methods available to study it are limited and not easily accessible. In this chapter, we detail a step-by-step method to systematically and quantitatively investigate glycosylation using fl uorescent lectin staining, following high-throughput RNAi-based downregulation of gene activities. We also provide a workfl ow for downstream analysis of the data generated.