title: Development of a novel telomerase assay using the PPDK-luciferin-luciferase detection system.
abstract: Telomerase participates in malignant transformation or immortalization of cells, and has attracted attention as an anticancer drug screening and diagnostic tumor marker. We developed a novel telomerase assay called the PPDK-luciferin-luciferase system bioluminescence assay (PLLBA) using pyruvate phosphate dikinase (PPDK). In this assay, pyrophosphate produced by the telomerase reaction and polymerase chain reaction (PCR) is converted to ATP by PPDK, and ATP is detected by the firefly luciferin-luciferase reaction. In this work, telomerase substrate was obtained in accordance with the telomeric repeat amplification protocol (TRAP). Telomerase-positive (500 cells/assay), -inactive (heated for 10 min at 85 °C) and -negative (only Chaps lysis buffer) samples were used. As a result, the findings clearly showed that the signal-to-noise (S/N) ratio of the positive cells was 39.5. After the telomerase reaction and PCR, PLLBA was completed ~ 120 s later. A high level of reproducibility was obtained with - coefficient of variation (CV) of 4.1% (positive cells). The detection limit for cells using telomerase was one cell per assay. This assay for telomerase activity was also shown to be adaptable to human cancer-derived cell lines.
authors.affiliation: Department of Analytical Biochemistry, School of Pharmacy, Showa University, Tokyo, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Karasawa
authors.forename: Y
authors.initials: Y
authors.lastname: Sano
authors.forename: H
authors.initials: H
authors.lastname: Arakawa
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D049454:Firefly Luciferin / Q000032:analysis*; D006801:Humans; D057230:Limit of Detection; D008156:Luciferases / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019098:Telomerase / Q000032:analysis*
chemical_list: D017931:DNA Primers; D049454:Firefly Luciferin; D008156:Luciferases; D019098:Telomerase
keywords: bioluminescence; diagnostic assay; pyrophosphate; pyruvate phosphate dikinase; telomerase; telomeric repeat; tumor marker