title: Flow cytometry-based functional selection of RNA interference triggers for efficient epi-allelic analysis of therapeutic targets.
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.
  
  
  CONCLUSIONS
  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.
authors: David R
authors: DR
authors: Micklem
authors: Magnus
authors: M
authors: Blø
authors: Petra
authors: P
authors: Bergström
authors: Erlend
authors: E
authors: Hodneland
authors: Crina
authors: C
authors: Tiron
authors: Torill
authors: T
authors: Høiby
authors: Christine
authors: C
authors: Gjerdrum
authors: Ola
authors: O
authors: Hammarsten
authors: Department of Biomedicine, University of Bergen, N-5009 Bergen, Norway. jim.lorens@biomed.uib.no.
authors: James B
authors: JB
authors: Lorens
mesh_terms: D000483:Alleles; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D015870:Gene Expression / Q000528:radiation effects; D015723:Gene Library; D018922:HL-60 Cells; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D016011:Normal Distribution; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011839:Radiation, Ionizing; D017422:Sequence Analysis, DNA; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53