Optimization of siRNA Knockdown in EGFP-Expressing Cell Lines Using High-Content Analysis
This protocol was adapted from “High-Content and High-Throughput Screening,” Chapter 13, in Cell Imaging (ed. Stephens). Scion Publishing Ltd., Oxfordshire, UK, 2006.INTRODUCTION
RNA interference using siRNA libraries is a powerful technology for elucidating gene function by downregulating gene expression at the post-transcriptional level. Phenotypic changes associated with siRNA knockdown can be monitored using cell lines expressing fluorescent reporter proteins. Test siRNAs are transiently transfected into the reporter cell line and behavior of the fluorescent reporter probe is monitored using a high-content imaging system. A range of cell features and additional fluorescent probes can be monitored to assess the effects of knockdown. This article describes how to optimize siRNA knockdown in EGFP-expressing cell lines.
