title: Detection of telomerase activity using microchip electrophoresis.
abstract: Telomerase participates in malignant transformation or immortalization of cells and thus has attracted attention as an anticancer drug target and diagnostic tumor marker. The telomeric repeat amplification protocol (TRAP) and improved TRAP methods (TRAP-fluorescence, TRAP-hybridization, etc.) are widely used forms of this telomerase assay. However, these approaches generally employ acrylamide gel electrophoresis after amplification of telomeric repeats by polymerase chain reaction (PCR), making these TRAP methods time consuming and technically demanding. In this study we developed a novel telomerase assay using microchip electrophoresis for rapid and highly sensitive detection of telomerase activity in cancer cells. The mixed gel of 0.8% hydroxypropyl methylcellulose (HPMC) and 0.3% polyethylene oxide (PEO) with SYBR Gold (fluorescent reagent) was used for microchip electrophoresis. As a result, the product amplified by a telomerase-positive cell could be measured in one cell per assay and detected with high reproducibility (CV=0.67%) in the short time of 100s.
authors.affiliation: Department of Analytical Biochemistry, School of Pharmacy, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Karasawa
authors.affiliation: Department of Analytical Biochemistry, School of Pharmacy, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555, Japan. Electronic address: arakawa@pharm.showa-u.ac.jp.
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Arakawa
mesh_terms: D045744:Cell Line, Tumor; D046209:Electrophoresis, Microchip / Q000379:methods*; D057075:Enzyme Assays / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D012680:Sensitivity and Specificity; D019098:Telomerase / Q000737:chemistry* / Q000235:genetics; D016615:Telomere / Q000737:chemistry
chemical_list: D019098:Telomerase
keywords: Diagnostic assay; Hydroxypropyl methylcellulose; Microchip electrophoresis; Polyethylene oxide; Telomerase; Tumor marker