Facile assay of telomerase activity utilizing a DNA-detectable chemiluminogenic reagent.
Telomerase shows increased activity in most human cancers and germ line cells, but not in normal human somatic cells. We describe a novel chemiluminescence method for the facile assay of telomerase activity in human cells. The telomerase substrate was incubated with the cell lysate containing various amounts of telomerase, and then the telomerase product was amplified by the polymerase-chained reaction (PCR). The PCR products were separated from the excess substrate, primer and deoxyribonucleotide triphosphates by a centrifugal filter, which distinguished different molecular sizes. The isolated products were reacted with a DNA-detectable chemiluminogenic reagent, 3,4,5-trimethoxyphenylglyoxal. The proposed assay method gave linearity for the telomerase activity in 100 to 10000 cells (r2=0.997), and allowed the assay not only of lower activity, but also of higher activity of telomerase without the requirement of any special labeled-PCR primers in the assay system.
Tonooka
Keiko
K
Faculty of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
Kabashima
Tsutomu
T
Shibata
Takayuki
T
Tang
Chenhong
C
Yu
Zhiqiang
Z
Kai
Masaaki
M
D045744:Cell Line, Tumor; D002498:Centrifugation; D004247:DNA / Q000032:analysis*; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D016133:Polymerase Chain Reaction; D019098:Telomerase / Q000032:analysis*
D004247:DNA; D019098:Telomerase