title: A high-throughput assay to identify modifiers of premature chromosome condensation.
abstract: Premature chromosome condensation (PCC) is a consequence of early mitotic entry, where mitosis begins before completion of DNA replication. Previously we have identified mutations in MCPH1, a DNA damage response and potential tumor suppressor gene, as a cause of primary microcephaly and PCC. Here we describe a high-throughput assay to identify modifiers of PCC. Reverse transfection of control siRNA followed by a forward transfection of MCPH1 small interfering RNA (siRNA) was performed to induce PCC. Condensin II subunits CAPG2 and CAPH2 were validated as PCC modifiers and therefore positive controls. Cell nuclei were detected by DAPI staining using an Operetta imaging system. PCC and nuclei number were determined using Columbus analysis software. Two batches of nine plates were used to determine assay efficacy. Each plate contained four negative (nontargeting) and eight positive control siRNAs. Mean % PCC was 12.35% (n = 72) for negative controls and 4.25% (n = 144) for positive controls. Overall false-positive and false-negative rates were 0% (n = 72) and 2.1% (n = 144), respectively. This assay is currently being used to screen a human druggable genome siRNA library to identify novel therapeutic targets for cancer treatment. The assay can also be used to identify novel compounds and genes that induce PCC.
authors: 1BioScreening Technology Group, Biomedical Health Research Centre, St. James's University Hospital, Beckett Street, Leeds, UK.
authors: Matthew
authors: M
authors: Adams
authors: Victoria J
authors: VJ
authors: Cookson
authors: Julie
authors: J
authors: Higgins
authors: Heather L
authors: HL
authors: Martin
authors: Darren C
authors: DC
authors: Tomlinson
authors: Jacquelyn
authors: J
authors: Bond
authors: Ewan E
authors: EE
authors: Morrison
authors: Sandra M
authors: SM
authors: Bell
mesh_terms: D045744:Cell Line, Tumor; D002875:Chromosomes / Q000187:drug effects*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D015870:Gene Expression; D057166:High-Throughput Screening Assays; D006801:Humans; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging; D034741:RNA, Small Interfering / Q000235:genetics; D015203:Reproducibility of Results; D014162:Transfection
chemical_list: D034741:RNA, Small Interfering
keywords: MCPH1; premature chromosome condensation; reverse genetics; screen