Faculty of Hepato-Pancreato-Biliary Surgery, Chinese PLA General Hospital, Beijing, China.
Medical school of Chinese PLA, Beijing, China.
J Clin Lab Anal. 2021 Jun;35(6):e23800. doi: 10.1002/jcla.23800. Epub 2021 May 7.
Telomerase is a ribonucleoprotein enzyme responsible for maintenance of telomere length which expressed in more than 85% of cancer cells but undetectable in most normal tissue cells. Therefore, telomerase serves as a diagnostic marker of cancers. Two commonly used telomerase activity detection methods, the telomerase repeated amplification protocol (TRAP) and the direct telomerase assay (DTA), have disadvantages that mainly arise from reliance on PCR amplification or the use of an isotope. A safe, low-cost and reliable telomerase activity detection method is still lacking.
We modified DTA method using biotin-labeled primers (Biotin-DTA) and optimized the method by adjusting cell culture temperature and KCl concentration. The sensitivity of the method was confirmed to detect endogenous telomerase activity. The reliability was verified by detection of telomerase activity of published telomerase regulators. The stability was confirmed by comparing the method with TRAP method.
Cells cultured in 32°C and KCl concentration at 200 mM or 250 mM resulted in robust Biotin-DTA signal. Endogenous telomerase activity can be detected, which suggested an similar sensitivity as DTA using radioactive isotope markers. Knockdown of telomerase assembly regulator PES1 and DKC1 efficiently reduced telomerase activity. Compared with TRAP method, Biotin-DTA assay offers greater signal stability over a range of analyte protein amounts.
Biotin-labeled, PCR-free, and nonradioactive direct telomerase assay is a promising new method for the easy, low-cost, and quantitative detection of telomerase activity.
端粒酶是一种核糖核蛋白酶，负责维持端粒长度，它在超过 85%的癌细胞中表达，但在大多数正常组织细胞中无法检测到。因此，端粒酶是癌症的诊断标志物。两种常用的端粒酶活性检测方法，端粒酶重复扩增协议（TRAP）和直接端粒酶检测（DTA），主要由于依赖于 PCR 扩增或使用同位素而存在缺点。仍然缺乏一种安全、低成本和可靠的端粒酶活性检测方法。
我们使用生物素标记的引物（Biotin-DTA）修改了 DTA 方法，并通过调整细胞培养温度和 KCl 浓度来优化该方法。该方法的灵敏度通过检测内源性端粒酶活性得到证实。通过检测已发表的端粒酶调节剂的端粒酶活性来验证该方法的可靠性。通过与 TRAP 方法进行比较来确认该方法的稳定性。
在 32°C 和 KCl 浓度为 200mM 或 250mM 的条件下培养的细胞产生了强烈的 Biotin-DTA 信号。可以检测到内源性端粒酶活性，这表明与使用放射性同位素标记物的 DTA 具有相似的灵敏度。敲低端粒酶组装调节剂 PES1 和 DKC1 可有效降低端粒酶活性。与 TRAP 方法相比，Biotin-DTA 测定在分析物蛋白量范围内提供了更大的信号稳定性。
无 PCR、非放射性的生物素标记直接端粒酶检测是一种很有前途的新方法，用于端粒酶活性的简便、低成本和定量检测。
