title: A 1,536-well ultra-high-throughput siRNA screen to identify regulators of the Wnt/beta-catenin pathway.
abstract: High-throughput siRNA screens are now widely used for identifying novel drug targets and mapping disease pathways. Despite their popularity, there remain challenges related to data variability, primarily due to measurement errors, biological variance, uneven transfection efficiency, the efficacy of siRNA sequences, or off-target effects, and consequent high false discovery rates. Data variability can be reduced if siRNA screens are performed in replicate. Running a large-scale siRNA screen in replicate is difficult, however, because of the technical challenges related to automating complicated steps of siRNA transfection, often with multiplexed assay readouts, and controlling environmental humidity during long incubation periods. Small-molecule screens have greatly benefited in the past decade from assay miniaturization to high-density plates such that 1,536-well nanoplate screenings are now a routine process, allowing fast, efficient, and affordable operations without compromising underlying biology or important assay characteristics. Here, we describe the development of a 1,536-well nanoplate siRNA transfection protocol that utilizes the instruments commonly found in small-molecule high throughput screening laboratories. This protocol was then successfully demonstrated in a triplicate large-scale siRNA screen for the identification of regulators of the Wnt/beta-catenin pathway.
authors.affiliation: Department of Automated Biotechnology, Merck & Co., North Wales, Pennsylvania, USA. namjin.chung@bms.com
authors.forename: Namjin
authors.initials: N
authors.lastname: Chung
authors.forename: Shane
authors.initials: S
authors.lastname: Marine
authors.forename: Emily A
authors.initials: EA
authors.lastname: Smith
authors.forename: Robert
authors.initials: R
authors.lastname: Liehr
authors.forename: S Todd
authors.initials: ST
authors.lastname: Smith
authors.forename: Louis
authors.initials: L
authors.lastname: Locco
authors.forename: Edward
authors.initials: E
authors.lastname: Hudak
authors.forename: Anthony
authors.initials: A
authors.lastname: Kreamer
authors.forename: Alison
authors.initials: A
authors.lastname: Rush
authors.forename: Brian
authors.initials: B
authors.lastname: Roberts
authors.forename: Michael B
authors.initials: MB
authors.lastname: Major
authors.forename: Randall T
authors.initials: RT
authors.lastname: Moon
authors.forename: William
authors.initials: W
authors.lastname: Arthur
authors.forename: Michele
authors.initials: M
authors.lastname: Cleary
authors.forename: Berta
authors.initials: B
authors.lastname: Strulovici
authors.forename: Marc
authors.initials: M
authors.lastname: Ferrer
mesh_terms: D000465:Algorithms; D000818:Animals; D002478:Cells, Cultured; D003627:Data Interpretation, Statistical; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation*; D015723:Gene Library; D006801:Humans; D008904:Miniaturization; D034741:RNA, Small Interfering / Q000494:pharmacology* / Q000627:therapeutic use; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured; D051153:Wnt Proteins / Q000235:genetics / Q000502:physiology*; D051176:beta Catenin / Q000235:genetics / Q000502:physiology*
chemical_list: D034741:RNA, Small Interfering; D051153:Wnt Proteins; D051176:beta Catenin