Abstract
The expression of genes is frequently manipulated in cell lines to study their cellular functions. The use of exogenous small Interfering RNAs (siRNAs) is a very efficient technique to temporarily downregulate the expression of genes of interest [reviewed by Hannon and Rossi (2004)]. A genome-wide siRNA library allows the user to study both the effect of each individual gene on a particular cell phenotype in a high throughput manner and also assess its phenotypic effect relative to all other genes targeted. Several factors that potentially influence the outcome of a screen need to be considered when performing a large siRNA screen (Jiang et al., 2011). Here we present a detailed protocol for a genome-wide screen to identify genes involved in anti-cancer drug resistance using the human siGENOME library from Dharmacon. In this protocol, we focus on resistance to treatment with the Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor (EGFR-TKI) erlotinib in the lung cancer cell line PC9, which is exquisitely sensitive to EGFR-TKIs (de Bruin et al., 2014). This protocol can be used for other cell lines and other drug treatments, as we expand in the Notes below.
Materials and Reagents
Equipment
Important considerations
Before performing a large-scale siRNA screen, there are a few crucial points to consider and to optimize in order to obtain the best results (Jiang et al., 2011). These include:
Details of these optimization steps are provided in the Notes section at the end of this protocol. We recommend performing, if possible, a pilot screen with a range of random siRNAs and selected control RNAs to determine the feasibility and screen read-out prior to the genome-wide screen.
Procedure
Notes
Acknowledgments
This protocol was developed for a study aiming to identify novel mechanisms of erlotinib resistance (de Bruin et al., 2014). E. B was funded by a fellowship from the Dutch Cancer Society and the High Throughput Screening facility at the Cancer Research UK-London Research Institute is funded by Cancer Research UK.
References
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