A Robust siRNA Screening Approach with Optimized Conditions for Large-Scale Transfection in Multiple Human Cancer...

During the past decades, advances in RNA interference (RNAi) technology have paved the way for the systematic exploration of gene function, and phenotypic screening of small interfering RNA (siRNA) oligonucleotides is a strategy still commonly pursued for the identification and validation of targets, particularly in oncology drug discovery. Here we present a method for large-scale automated siRNA transfection and cell phenotypic screening using colony formation as a readout. Experimental conditions were optimized to achieve efficient and nontoxic transfection of siRNA oligonucleotides in different cell lines using liposomal reagents. For each gene, the most active and specific siRNA oligos were selected through a phenotypic prescreening in HeLa cells, selected as control cell line, and grouped in the same oligo pool. Cells were then transfected at low seeding density in 96-well plates, and after 7-14 days colony formation was analyzed. We have found this procedure to be more sensitive than standard 48-72 h proliferation assays for identifying genes essential for cell viability/proliferation, as it allows to reveal long-term consequences in slow growing cell lines, or phenotypes that occur after multiple cell divisions. This approach generated robust and reliable results through the limitation of siRNA off-target toxic effects by combining a pool of different siRNA oligos designed against the same target. Furthermore, a parallel evaluation of gene silencing phenotypes is performed against a large panel of cell lines, allowing the simultaneous identification of target related genetic dependencies in several cancer cell line models of different tumor origin.