A quantitative assay of telomerase activity

Purpose Telomerase is a ribonucleoprotein that extends telomeres at the end s of chromosome. Increased telomerase activity is associated with cellular immortality. The currently available assay for telomerase, ie., telomeric r epeat amplification protocol (TRAP), consists of 2 steps: (a) telomerase-me diated extension of an oligonucleotide primer by the enzyme-containing extr acts of cells and tissues, and (b) amplification of the telomerase-extended primer products by polymerase chain reaction (PCR) and detection of the PC R products. It is generally accepted that the current TRAP assay lacks quan titative precision. The present study was to develop a quantitative telomer ase 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 extract ion 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 (r(2) = 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 conven tional 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 int er- and intra-day variability (>3-fold), and (c) showed a 2 to 4-fold broad er range of Linearity in the standard curve. The higher assay sensitivity f urther enabled the use of a nonradioactive method, i.e., ethidium bromide s taining of DNA, to detect the TRAP products, as opposed to the use of radio active 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.