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Open Access Methodology article

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

David R Micklem12, Magnus Blø12, Petra Bergström3, Erlend Hodneland1, Crina Tiron1, Torill Høiby1, Christine Gjerdrum1, Ola Hammarsten3 and James B Lorens1*

Author Affiliations

1 Department of Biomedicine, University of Bergen, N-5009 Bergen, Norway

2 BerGenBio A/S, N-5009, Bergen, Norway

3 Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden

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BMC Biotechnology 2014, 14:57  doi:10.1186/1472-6750-14-57

Published: 21 June 2014

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.