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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.

In cells treated with TNFalpha, NF-kappaB translocates from its predominately cytoplasmic localization to become concentrated in nuclei where it interacts with DNA as a transcription factor (Figure 1). In cells transfected with TNFR siRNA, the translocation of NF-kappaB in response to TNFalpha treatment was dramatically reduced.

Figure 1. (Figure 4 from the print edition of TechNotes) Reduction in TNFalpha-induced NF-kappaB Translocation Caused By an siRNA Targeting the TNFalpha Receptor. HeLa cells were transfected with siRNAs targeting IL-1 (Control siRNA) and the TNFalpha receptor (TNFR siRNA). Forty eight hours post-transfection, the cells were recovered from the 24 well plates in which they were transfected and transferred to wells of a 96 well plate. Following overnight recovery, the cells were processed for immunofluorescence staining of NF-kappaB.

There are several distinct signaling pathways that can trigger the nuclear translocation of NF-kappaB. This suggests that RNAi could be used to distinguish activators of these pathways. As shown in Figure 2, while the cell population transfected with TNFR siRNA showed significantly reduced response to TNFalpha treatment (asterisk), the same cell population still responded robustly to IL1alpha treatment. This clearly shows the specificity of the siRNA transfection and highlights the applicability to drug discovery.

Figure 2. Nuclear Translocation of NF-kappaB in Response to 10 ng/mL TNFalpha or IL1alpha in Untreated or TNFR siRNA Transfected HeLa Cells. Nuclear translocation was rea dily quantitated using Cellomics' Cytoplasm to Nucleus Translocation BioApplication for the ArrayScan HCS Reader.

1. Giuliano KA, Haskins JR, Taylor DL. (2003) Advances in High Content Screening for Drug Discovery. ASSAY Drug Develop Technol (In Press).

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