A Quantitative Method to Measure Telomerase Activity by Bioluminescence Connected with Telomeric Repeat Amplification Protocol

Telomerase is expected to be a new biomarker for cancer diagnosis. The telomeric repeat amplification protocol (TRAP) is a sensitive method to detect telomerase activity. However, TRAP and its modified protocols are not always suitable for measuring telomerase activity of a large number of clinical...

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Bibliographic Details
Published in:Analytical biochemistry 2001-12, Vol.299 (2), p.188-193
Main Authors: Xu, Shunqing, He, Min, Yu, Hongping, Cai, Xiaokun, Tan, Xianglin, Lu, Bin, Shu, Baihua
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - analysis
bioluminescence
Carcinoma - enzymology
Deoxyadenine Nucleotides - chemistry
Female
Humans
Luciferases - analysis
Luminescent Measurements
Polymerase Chain Reaction - methods
pyrophosphate
Repetitive Sequences, Nucleic Acid
Sensitivity and Specificity
telomerase
Telomerase - analysis
Telomere - genetics
TRAP assay
Tumor Cells, Cultured
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Summary:Telomerase is expected to be a new biomarker for cancer diagnosis. The telomeric repeat amplification protocol (TRAP) is a sensitive method to detect telomerase activity. However, TRAP and its modified protocols are not always suitable for measuring telomerase activity of a large number of clinical samples to diagnosis cancer because these methods generally require a time-consuming detection step such as gel electrophoresis. To improve the procedure for mass diagnosis, we applied bioluminescence to replace the detection step. Telomerase activity is measured by evaluating the amount of inorganic pyrophosphate generated in PCR amplification of telomerase elongation product, with use of the sensitive enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA). TRAP connected with ELIDA (TRAP–ELIDA) can quantitatively detect telomerase activity within linearity from 2 to 1000 cell equivalents. The ELIDA signals accorded with results of TRAP–SYBR green staining, and the results of ELIDA were significantly correlated to those of TRAP connected with an enzyme-linked immunosorbent assay (TRAP–ELISA) (r2 = 0.992, P < 0.001). TRAP–ELIDA is a simple and sensitive method to quantify telomerase activity without time-consuming gel electrophoresis. Because TRAP–ELIDA measures telomerase activity with a luminometer, it could be applied to a large number of clinical samples at the same time.
ISSN:0003-2697
1096-0309
DOI:10.1006/abio.2001.5418