title: High-throughput RNA interference screening: tricks of the trade.
abstract: The process of validating an assay for high-throughput screening (HTS) involves identifying sources of variability and developing procedures that minimize the variability at each step in the protocol. The goal is to produce a robust and reproducible assay with good metrics. In all good cell-based assays, this means coefficient of variation (CV) values of less than 10% and a signal window of fivefold or greater. HTS assays are usually evaluated using Z' factor, which incorporates both standard deviation and signal window. A Z' factor value of 0.5 or higher is acceptable for HTS. We used a standard HTS validation procedure in developing small interfering RNA (siRNA) screening technology at the HTS center at Southern Research. Initially, our assay performance was similar to published screens, with CV values greater than 10% and Z' factor values of 0.51 ± 0.16 (average ± standard deviation). After optimizing the siRNA assay, we got CV values averaging 7.2% and a robust Z' factor value of 0.78 ± 0.06 (average ± standard deviation). We present an overview of the problems encountered in developing this whole-genome siRNA screening program at Southern Research and how equipment optimization led to improved data quality.
authors.affiliation: High Throughput Screening Center, Southern Research Institute, Birmingham, AL, USA. nebane@southernresearch.org
authors.forename: N Miranda
authors.initials: NM
authors.lastname: Nebane
authors.forename: Tatjana
authors.initials: T
authors.lastname: Coric
authors.forename: Kanupriya
authors.initials: K
authors.lastname: Whig
authors.forename: Sara
authors.initials: S
authors.lastname: McKellip
authors.forename: Lakeisha
authors.initials: L
authors.lastname: Woods
authors.forename: Melinda
authors.initials: M
authors.lastname: Sosa
authors.forename: Russell
authors.initials: R
authors.lastname: Sheppard
authors.forename: Lynn
authors.initials: L
authors.lastname: Rasmussen
authors.forename: Mary-Ann
authors.initials: MA
authors.lastname: Bjornsti
authors.forename: E Lucile
authors.initials: EL
authors.lastname: White
mesh_terms: D000818:Animals; D005820:Genetic Testing / Q000295:instrumentation / Q000379:methods*; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000592:standards; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D015203:Reproducibility of Results
chemical_list: D034741:RNA, Small Interfering
keywords: RNA interference; coefficient of variation; high throughput screening; small interfering RNA