Journal of Biotechnology 129 (2007) 352–365 An RNAi screening platform to identify secretion machinery in mammalian cells Jeremy C. Simpson a,∗ , Cihan Cetin a , Holger Erfle a , Brigitte Joggerst a , Urban Liebel b , Jan Ellenberg b , Rainer Pepperkok a a Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany b Gene Expression Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany Received 14 August 2006; received in revised form 7 December 2006; accepted 22 December 2006 Abstract Integrative approaches to study protein function in a cellular context are a vital aspect of understanding human disease. Genome sequencing projects provide the basic catalogue of information with which to unravel gene function, but more systematic applications of this resource are now necessary. Here, we describe and test a platform with which it is possible to rapidly use RNA interference in cultured mammalian cells to probe for proteins involved in constitutive protein secretion. Synthetic small interfering RNA molecules are arrayed in chambered slides, then incubated with cells and an assay for secretion performed. Automated microscopy is used to acquire images from the experiments, and automated single-cell analysis rapidly provides reliable quantitative data. In test arrays of 92 siRNA spots targeting 37 prospective membrane traffic proteins, our approach identifies 7 of these as being important for the correct delivery of a secretion marker to the cell surface. Correlating these findings with other screens and bioinformatic information makes these candidates highly likely to be novel membrane traffic machinery components. © 2007 Elsevier B.V. All rights reserved. Keywords: RNA interference; Secretory membrane traffic; High content screening microscopy 1. Introduction Two recent events provide the cornerstone of an emerging strategy as to how we might systematically ∗ Corresponding author. Tel.: +49 6221 387 0; fax: +49 6221 387 8306. E-mail addresses: simpson@embl.de (J.C. Simpson), pepperko@embl.de (R. Pepperkok). study cell function. The first is the sequencing of the human genome and its continually improving annota- tion, thereby providing the basic catalogue of all human proteins (Rual et al., 2004). The second key event is the ability to specifically and rapidly silence individual genes and accordingly downregulate their correspond- ing proteins in mammalian cells through the use of RNA interference (RNAi) (reviewed by Novina and Sharp, 2004). Although targeted gene ablation has long 0168-1656/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2006.12.027