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Using Validated siRNAs in Functional Genomic Assays


Collections of siRNAs targeting key human genes are ideal for studying protein function in cells. To identify genes involved in a given cellular process, cells can be transfected with different siRNAs and assayed for distinct response profiles such as cell cycle arrest. Cellomics, Inc., who pioneered the field of High Content Screening (HCS), has built cellular response profiles for several of Ambion's Silencer Validated siRNAs, utilizing their automated cell-based screening platform (1). For example, a Silencer Validated siRNA targeting the TNFalpha receptor (TNFR) can be used to identify gene products associated with TNFalpha-induced activation of the NF-kappaB pathway. Upon activation, NF-kappaB undergoes a translocation from its normal localization in the cytoplasm to the nucleus. Cellomics ArrayScan HCS Reader can be utilized to rapidly quantitate such nuclear translocation events. In the experiment detailed below, TNFalpha-triggered activation of NF-kappaB nuclear translocation was used as a functional HCS assay of TNFR modulation. When TNFR expression level was reduced, NF-kappaB nuclear translocation was expected to be suppressed or reduced. On the other hand, NF-kappaB nuclear translocation triggered with ligands distinct from TNFalpha was expected to occur normally. Thus, a functional assay for the level of TNFR protein in the cell, which did not rely on direct measurement of TNFR protein content, was created.
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