Analysis of a Time-resolved, siRNA High Content Screen Clusters New NFkB Regulators to Known Pathway

October 15, 2009

Presented at MipTec, Basel, Switzerland

The gastric pathogen Helicobacter pylori is responsible for gastric inflammation and the second highest number of infection-associated cancers. The transcription factor NFkB, a key mediator of host tissue inflammation, induces the expression of pro-inflammatory cytokines, which have been shown to be important for the development of gastric cancer. To better understand NFkB activation in response to H. pylori infection, a genome wide RNA interference screen was performed, resulting in 300 primary hits. To characterize the effects of these 300 hits a time-series high content screen (HCS) with three inducers (H. pylori, IL-1b and TNFa) was carried out. Proteins specifically affecting the cellular response to one of the inducers, as well as those which were common to all inducers, were identified.

Data analysis was performed on Genedata Screener® and Genedata AnalystTM software platforms. The effect of three different inducers on almost 40 different features extracted from the HCS images were analyzed, each measured over nine time points in triplicates (about 5 Million data points in total). Data standardization, feature selection and replicate condensing were optimized for this complex  experimental design. Time-dependent profiles of {siRNA x inducer} combinations were clustered to yield groups of genes showing similar outcomes on siRNA silencing. The results were interpreted in the context of known inflammatory pathways.

In summary, a general protocol for cross-experimental analysis of time-dependent HCS data was devised, focusing on detailed comparison of siRNA silencing profiles in large HCS data sets. Targeted genes of known protein function allowed further genes involved in these pathways to be found, improving understanding of the processes leading to inflammation after H. pylori infection.

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