Fred Hutchinson Cancer Research Center (FHCRC1): Functional Exploration of the Druggable Genome in MYCN Amplified and Non-amplified Neuroblastoma
To identify candidate drugs targets for neuroblastoma with MYCN amplification we performed parallel siRNA screens with a druggable genome collection of ~6,700 genes comparing MYCN amplified and non-MYCN amplified cell lines: SK-N-BE2 (MYCN amplified) and SK-N-AS (non amplified). The Hits from each cell lines were determined based on their significance with respect to their differential activity in the presence or absence of RA within each cell line. Hits for each cell line were also ranked according to their P-value, based on the three replicates. Integration with gene expression data from several databases also allowed us to pinpoint genes whose expression is selective for MYCN amplified neuroblastoma (this analysis was carried out in collaboration with the CTD2 group at Columbia University, led by Dr. Califano).
Neuroblastoma cell lines, SK-N-BE2 (MYCN-amplified) and SK-N-AS (non-amplified) were obtained from the American Tissue Type Collection (ATCC) and cultured in RPMI with 10% FBS and Penicillin and Streptomycin at the concentration recommended. 25mM HEPES was added during the screens to minimize variation of pH during handling of the plates.
High-throughput siRNA transfections
Transfection feasibility for each cell line was established using a factorial-based optimization scheme with variable parameters in cell density, liposomal, and siRNA concentration. All transfection conditions were statistically evaluated using a simple Z-factor score to determine differentials between mock-transfected cell (transfection reagent only) versus a positive control for growth inhibition (KIF11) and a negative control (non-targeting siRNA). Based on these preliminary experiments, the optimal transfection condition was selected for each cell line as following: cells were seeded at 500 cells/well; the next day 2.5 μl of Dharmafect I (6.25 μl/ml) and siRNA (1.25 pM/well) mix was added and viability with CellTiter-Glo was read at 96 hrs. The siRNA library targeting the druggable genome (~6,700 genes) was tested in triplicates to establish experimental variability and statistical validity. A detailed description of the method is available in the supplementary information by Toyoshima et al.
The siRNA library targeting the druggable genome (MISSION® siRNA Human Druggable Genome, Sigma) was tested with viability as the phenotypic endpoint. Cell viability was quantified using an Envision Multilabel detector/plate reader (PerkinElmer) with the CellTiter-Glo assay (Promega) that measures ATP concentrations in cell lysates. siRNA libraries utilized pools of 3 independent siRNAs targeting each gene, in a one gene per well approach. RNAi screens were performed in 384-well format in triplicate, in independent plates, utilizing robotics instrumentation available at the University of Washington - Quellos facility.
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