Protocol

Analysis of siRNA Knockdown of Cell-Cycle Control Genes in G₁/S and G₂/M Cell-Cycle Phase Marker Cell Lines Using Multiplexed 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 provides a method of screening an siRNA cell-cycle array using cell lines that report cell-cycle phase. The subcellular distribution and intensity of the fluorescent phase reporter changes in a cell-cycle-dependent manner. The phase marker assay is multiplexed with an assay for bromodeoxyuridine (BrdU) incorporation, which identifies cells that have undergone DNA replication. When a G₂/M cell-cycle phase reporter cell line is multiplexed with BrdU incorporation, information from both assays enables assignment of each cell to one of five distinct phases of the cell cycle (G₁, S, G₂, prophase, or mitosis).