We have implemented a fully functional pipeline optimized to carry out RNAi-based high-throughput imaging screens in fixed and living mammalian cells. The pipeline consists of several commercially available and custom-designed RNAi libraries, a robotic workflow for RNAi transfection, automated immunofluorescence detection, high-content and high-throughput imaging using the PE OPERA microscopy platform, and several image analysis/informatics systems, including commercial PE Acappella software and custom designed software. We have validated the workflow by performing RNAi screens using several novel in vivo assays including protein degradation, subcellular protein localization and kinetic assays. The assay development projects have confirmed that the high-content screening pipeline can quantify significant changes in specific subcellular structures, which can most accurately be measured with high-resolution imaging methods. We have initiated 14 collaborative projects, and completed our contribution to six of these, moving the projects back to the initiating laboratories for follow-up studies. The HiTIF is prepared to collaborate with NCI laboratories and will continue to assist with sample preparation, high-throughput microscopy, and associated analysis.
