Optimized for routine: highly sensitive fluorescent Telomeric Repeat Amplification Protocol (f-TRAP)

The strict suppression of telomerase activity (TA) in terminally differentiated human cells causes a shortening of the chromosome ends after each cell division. This tumor suppression surveillance mechanism is associated with a limited number of cell divisions known as Hayflick limit. Here we present an optimized protocol for measuring TA that combines a fluorescently labeled bait primer and polymerase chain reaction (PCR) amplification with analytical capillary electrophoresis (CE) to achieve a detection limit of one telomerase-positive cell per ten thousand negative cells. In research laboratories today, a broad panel of TRAP assay protocols enables the assessment of the immortality of newly generated cell lines or the unambiguous evaluation of the reprogramming of induced pluripotent stem cells (iPSCs). The present f-TRAP protocol, optimized for routine use, enables fast ad hoc application for single measurements up to a high throughput of mass samples using a triplicate approach of different lysate concentrations. Final CE analysis facilitates standardized data processing and storage for documentation of results and could make f-TRAP a useful assay in research and clinical oncology.
Telomerase activity is the most universal of all known tumor markers and a promising target for anti-tumor therapies. The robustness and high sensitivity of f-TRAP can (i) demonstrate early validation of cell line immortality in the establishment of model systems, (ii) lead to rapid detection of successful reprogramming or differentiation of cells in vitro and (iii) be used in high throughput assays in the search for new inhibitors of telomerase activity.

在终末分化的人类细胞中严格抑制端粒酶活性（telomerase activity, TA），会导致每次细胞分裂后染色体末端逐渐缩短。这种肿瘤抑制监视机制与有限的细胞分裂次数相关，该次数被称为海弗利克极限（Hayflick limit）。本研究提出了一种优化的端粒酶活性检测方案，该方案将荧光标记的诱饵引物、聚合酶链式反应（PCR）扩增与分析型毛细管电泳（CE）相结合，可实现每10000个阴性细胞中检出1个端粒酶阳性细胞的检测限。如今，科研实验室中已存在多种端粒重复序列扩增法（TRAP assay）方案，可用于评估新构建细胞系的永生化状态，或是对诱导多能干细胞（iPSCs）的重编程过程进行精准评估。本研究优化的f-TRAP方案适配日常实验需求，可通过设置不同裂解液浓度的三次重复实验，实现单次检测的快速临时应用，乃至大规模样本的高通量检测。后续的CE分析可实现标准化的数据处理与结果存档，这使得f-TRAP有望成为肿瘤学研究与临床领域中极具应用价值的检测手段。
端粒酶活性是目前已知最具普遍性的肿瘤标志物之一，同时也是抗肿瘤治疗的极具潜力的靶点。f-TRAP具备优异的稳定性与极高的灵敏度，其应用场景包括：（i）在模型系统构建过程中，早期验证细胞系的永生化状态；（ii）快速检测体外细胞重编程或分化是否成功；（iii）用于高通量检测，以筛选新型端粒酶活性抑制剂。