A multiplexed siRNA screening strategy to identify genes in the PARP pathway.

Abstract

Gene silencing by RNA interference has become a powerful tool to help identify genes that regulate biological processes. However, the complexity of the biology probed and the incomplete validation of the reagents used make it difficult to interpret the results of genome-wide siRNA screens. To address this challenge and maximize the return on the efforts required for validating genomic screen hits, the screening strategy must be designed to increase the robustness of the primary screening hits and include assays that inform on the mechanism of action of the knocked-down transcripts. Here, we describe the implementation of a small interfering RNA (siRNA) screen to identify genes that sensitize the effect of poly-(ADP ribose)-polymerase (PARP) inhibitor on cell survival. In the strategy we designed for the primary screen, two biological activities, apoptosis and cell viability, were measured simultaneously at different time points in the presence and absence of a PARP inhibitor (PARPi). The multiplexed assay allowed us to identify PARPi sensitizers induced by both caspase-dependent and independent mechanisms. The multiplexed screening strategy yielded robust primary hits with significant enrichment for DNA repair genes, which were further validated using relevant high-content imaging assays and confirmation of transcript knockdown by real-time PCR (rtPCR).

DOI: 10.1177/1087057112453071

Cite this paper

@article{Stec2012AMS, title={A multiplexed siRNA screening strategy to identify genes in the PARP pathway.}, author={Erica M. Stec and Louis Locco and Stacey Szymanski and Steven R. Bartz and Carlo Toniatti and Rachel H. V. Needham and Anthony Palmieri and Michael O. Carleton and Michele A. Cleary and Aimee L. Jackson and Peter S. Linsley and Berta Strulovici and Marc Ferrer and Francesca Santini}, journal={Journal of biomolecular screening}, year={2012}, volume={17 10}, pages={1316-28} }