title: 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 fluorescent lectin staining, following high-throughput RNAi-based downregulation of gene activities. We also provide a workflow for downstream analysis of the data generated.
authors.affiliation: Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, 61 Biopolis Drive, Proteos, Singapore, 138673, Singapore.
authors.forename: Germaine Y
authors.initials: GY
authors.lastname: Goh
authors.forename: Frederic A
authors.initials: FA
authors.lastname: Bard
mesh_terms: D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D055785:Gene Knockdown Techniques; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D006801:Humans; D007091:Image Processing, Computer-Assisted; D037102:Lectins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D034622:RNA Interference
chemical_list: D002352:Carrier Proteins; D037102:Lectins