PURPOSE: Telomerase is a ribonucleoprotein that extends telomeres at the ends of chromosome. Increased telomerase activity is associated with cellular immortality. The currently available assay for telomerase, i.e., telomeric repeat amplification protocol (TRAP), consists of 2 steps: (a) telomerase-mediated extension of an oligonucleotide primer by the enzyme-containing extracts of cells and tissues, and (b) amplification of the telomerase-extended primer products by polymerase chain reaction (PCR) and detection of the PCR products. It is generally accepted that the current TRAP assay lacks quantitative precision. The present study was to develop a quantitative telomerase assay with greater precision and sensitivity. METHODS: This new method used the primer extension method as in TRAP, plus the following modifications: (a) used a lysis buffer that yielded complete lysis of nuclei; (b) removal of PCR inhibitors by phenol/chloroform extraction after primer extension; and (c) used primers for the internal standard that were designed to reduce their competition with the telomerase products for PCR. RESULTS: The modified method showed a good correlation (r2 = 0.99, P < 0.001) between telomerase amount (expressed as total protein in cell lysate) and its activity (expressed as telomerase products). Compared to the conventional TRAP, the new method (a) was more sensitive (average of 5.5-fold in cultured cancer cells and >5.9-fold in patient tumors), (b) had a lower inter- and intra-day variability (>3fold), and (c) showed a 2 to 4-fold broader range of linearity in the standard curve. The higher assay sensitivity further enabled the use of a nonradioactive method, i.e., ethidium bromide staining of DNA, to detect the TRAP products, as opposed to the use of radioactive nucleotide and the more labor-intensive autoradiography mandated by the conventional TRAP. CONCLUSION: We report here a quantitative assay for telomerase activity in cultured human cancer cells and patient tumors.
PURPOSE: Telomerase is a ribonucleoprotein that extends telomeres at the ends of chromosome. Increased telomerase activity is associated with cellular immortality. The currently available assay for telomerase, i.e., telomeric repeat amplification protocol (TRAP), consists of 2 steps: (a) telomerase-mediated extension of an oligonucleotide primer by the enzyme-containing extracts of cells and tissues, and (b) amplification of the telomerase-extended primer products by polymerase chain reaction (PCR) and detection of the PCR products. It is generally accepted that the current TRAP assay lacks quantitative precision. The present study was to develop a quantitative telomerase assay with greater precision and sensitivity. METHODS: This new method used the primer extension method as in TRAP, plus the following modifications: (a) used a lysis buffer that yielded complete lysis of nuclei; (b) removal of PCR inhibitors by phenol/chloroform extraction after primer extension; and (c) used primers for the internal standard that were designed to reduce their competition with the telomerase products for PCR. RESULTS: The modified method showed a good correlation (r2 = 0.99, P < 0.001) between telomerase amount (expressed as total protein in cell lysate) and its activity (expressed as telomerase products). Compared to the conventional TRAP, the new method (a) was more sensitive (average of 5.5-fold in cultured cancer cells and >5.9-fold in patienttumors), (b) had a lower inter- and intra-day variability (>3fold), and (c) showed a 2 to 4-fold broader range of linearity in the standard curve. The higher assay sensitivity further enabled the use of a nonradioactive method, i.e., ethidium bromide staining of DNA, to detect the TRAP products, as opposed to the use of radioactive nucleotide and the more labor-intensive autoradiography mandated by the conventional TRAP. CONCLUSION: We report here a quantitative assay for telomerase activity in cultured humancancer cells and patienttumors.
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