title: Rapid monitoring of RNA degradation activity in vivo for mammalian cells.
abstract: We have developed a rapid fluorescence assay based on fluorescence resonance energy transfer (FRET) for the monitoring of RNA degradation activity in mammalian cells. In this technique, double-stranded RNA (dsRNA) fluorescent probes are used. The dsRNA fluorescent probes consist of a 5' fluorophore-labeled strand hybridized to a 3' quencher-labeled strand, and the fluorescent dye is quenched by a quencher dye. When the dsRNA is degraded by nascent RNases in cells, the fluorescence emission of the fluorophore is induced following the degradation of the double strands. The degradation rates of the dsRNA are decelerated in response to chemical or environmental toxicity; therefore, in the case of cellular toxicity, the dsRNA is not degraded and remains intact, thus quenching the fluorescence. Unlike in conventional cell-counting assays, this new assay eliminates time-consuming steps, and can be used to simply evaluate the cellular toxicity via a single reaction. Our results demonstrate that this assay can rapidly quantify the RNA degradation rates in vivo within 4 h for three model chemicals. We propose that this assay will be useful for monitoring cellular toxicity in high-throughput applications.
authors.affiliation: Environmental Management Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba, Ibaraki 305-8569, Japan. Electronic address: h.tani@aist.go.jp.
authors.forename: Hidenori
authors.initials: H
authors.lastname: Tani
authors.affiliation: Environmental Management Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba, Ibaraki 305-8569, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Sato
authors.affiliation: Environmental Management Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba, Ibaraki 305-8569, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Torimura
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D015335:Molecular Probes / Q000032:analysis / Q000737:chemistry; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D020871:RNA Stability; D012330:RNA, Double-Stranded / Q000032:analysis / Q000737:chemistry; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D015335:Molecular Probes; D012330:RNA, Double-Stranded; D012313:RNA
keywords: Biosensors; Cell-based assay; Fluorescent probe; RNA degradation; Toxicity testing