Fred Hutchinson Cancer Research Center (FHCRC-1): Identification of Candidate Therapeutic Targets in Head and Neck Cancer Using Functional Kinomics
Kinome-wide siRNA screens targeting 713 human (MISSION® siRNA Human Gene Family Set, Sigma) were performed with viability as the phenotypic endpoint on five HNSCC lines: JHU-019; PCI15A and 15B; UM-SCC14A and 14C.
siRNA libraries were constructed and utilized in pools of 3 independent siRNAs targeting each gene, in a one gene per well approach. RNAi screens were performed in 384-well format utilizing robotics instrumentation available at the University of Washington - Quellos facility. Transfection feasibility of each cell line was established using a factorial optimization. Mock condition and a non-targeting universal siRNA control were utilized as negative controls, while a siRNA directed at KIF11 (kinesin-like protein), which arrests cells in mitosis was utilized as a positive control. All reagent conditions were statistically evaluated using a simple Z-factor score to evaluate differentials and variability of replicates (i.e. potent cell killing with KIF11 at the lowest toxicity possible in the mock universal controls) to select an optimized transfection condition for each cell line. All kinases were tested in triplicate to establish experimental variability and statistical validity. Scrambled siRNA negative controls were used to monitor dynamic range and off-target effects and the results were standardized to mock-transfected cells.
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