Flow cytometry-based functional selection of RNA interference triggers for efficient epi-allelic analysis of therapeutic targets

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Abstract

Background: The dose-response relationship is a fundamental pharmacological parameter necessary to determine therapeutic thresholds. Epi-allelic hypomorphic analysis using RNA interference (RNAi) can similarly correlate target gene dosage with cellular phenotypes. This however requires a set of RNAi triggers empirically determined to attenuate target gene expression to different levels.Results: In order to improve our ability to incorporate epi-allelic analysis into target validation studies, we developed a novel flow cytometry-based functional screening approach (CellSelectRNAi) to achieve unbiased selection of shRNAs from high-coverage libraries that knockdown target gene expression to predetermined levels. Employing a Gaussian probability model we calculated that knockdown efficiency is inferred from shRNA sequence frequency profiles derived from sorted hypomorphic cell populations. We used this approach to generate a hypomorphic epi-allelic cell series of shRNAs to reveal a functional threshold for the tumor suppressor p53 in normal and transformed cells.Conclusion: The unbiased CellSelectRNAi flow cytometry-based functional screening approach readily provides an epi-allelic series of shRNAs for graded reduction of target gene expression and improved phenotypic validation. © 2014 Micklem et al.; licensee BioMed Central Ltd.

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Micklem, D. R., Blø, M., Bergström, P., Hodneland, E., Tiron, C., Høiby, T., … Lorens, J. B. (2014). Flow cytometry-based functional selection of RNA interference triggers for efficient epi-allelic analysis of therapeutic targets. BMC Biotechnology, 14. https://doi.org/10.1186/1472-6750-14-57

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