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

BMC Biotechnol. 2014 Jun 21;14:57. doi: 10.1186/1472-6750-14-57.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Cell Line, Tumor
  • Flow Cytometry*
  • Gene Expression / radiation effects
  • Gene Library
  • HL-60 Cells
  • Human Umbilical Vein Endothelial Cells
  • Humans
  • Normal Distribution
  • RNA Interference*
  • RNA, Messenger / metabolism
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • Radiation, Ionizing
  • Sequence Analysis, DNA
  • Tumor Suppressor Protein p53 / antagonists & inhibitors
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism

Substances

  • RNA, Messenger
  • RNA, Small Interfering
  • TP53 protein, human
  • Tumor Suppressor Protein p53