University of Texas Southwestern Medical Center: High-throughput siRNA Screening of a Non-small Cell Lung Cancer (NSCLC) Cell Line Panel
The goal of this project is to use siRNA screens to identify NSCLC-selective siRNAs from two genome-wide libraries that will allow us to functionally define genetic dependencies of subtypes of NSCLC. Using bioinformatics tools, the CTD2 center at the University of Texas Southwestern Medical Center are discovering associations between this functional data (siRNAs) and NSCLC mutational status, methylation arrays, gene expression arrays, and copy number variation data that will help us identify new targets and enrollment biomarkers.
Two commercial genome wide siRNA libraries from Ambion (library 1; 21,585 genes) and Dharmacon (library 2; 18,175 genes) were purchased in the 96-well plate format. siRNAs were dissolved in 13 siRNA buffer (Dharmacon) overnight to a final concentration of 10 mM and stored at 80°C prior to use. Library 1 and 2 are mixes of 3 and 4 individual siRNA oligos/genes, respectively. For the primary and secondary screens, cells were cultured in ACL4 (normal cell lines and HCC4017) with 2% FBS or RPMI medium (all other cancer lines) with 5% FBS. For reverse transfections 10 pmole of each siRNA pool was transferred (3 ml) to serum free media (95 ml/well) in empty 96-well assay plates (Costar). Thirty μl of this siRNA solution was transferred to an empty 96-well optical assay plate (BioMek), incubated for 5 minutes, then mixed with 10 ml transfection reagent solution (0.13 ml RNAiMax [Invitrogen] in 10 ml serum free medium), and incubated for 15 minutes. Cells were harvested and diluted in parallel (10,000 cells/100 ml), then added to the siRNA-lipid mix and incubated for 96 hours. CellTiter-Glo (Promega) assays were performed using 15 ml reagent/well followed by a 10 minute incubation prior to quantitation of luminescence with an Envision plate reader (PerkinElmer). Primary screen data were normalized by top quantile per plate. Standard Deviations of the triplicate activities were calculated for each gene.
For questions, please contact Bruce Posner.