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

IF 2.2 4区 工程技术 Q3 BIOCHEMICAL RESEARCH METHODS
BioTechniques Pub Date : 2024-10-01 Epub Date: 2024-12-15 DOI:10.1080/07366205.2024.2432805
Silke Fähnrich, Anne Wedemann, Laura Steenpass, Wilhelm Gerhard Dirks
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引用次数: 0

Abstract

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.

常规优化:高灵敏度荧光端粒重复扩增协议(f-TRAP)。
在终末分化的人类细胞中,端粒酶活性(TA)受到严格抑制,导致每次细胞分裂后染色体末端缩短。这种肿瘤抑制监视机制与有限数量的细胞分裂有关,称为海弗利克限制。在这里,我们提出了一种优化的检测TA的方案,该方案结合了荧光标记诱饵引物和聚合酶链反应(PCR)扩增以及分析毛细管电泳(CE),以达到每10,000个端粒酶阳性细胞中有一个端粒酶阳性细胞的检测限。在今天的研究实验室中,广泛的TRAP检测方案能够评估新生成的细胞系的不朽性或对诱导多能干细胞(iPSCs)的重编程进行明确的评估。目前的f-TRAP协议针对常规使用进行了优化,可以快速地进行单次测量,使用不同裂解物浓度的三次方法对大量样品进行高通量测量。最终的CE分析促进了标准化的数据处理和结果记录的存储,并可以使f-TRAP成为研究和临床肿瘤学中有用的检测方法。
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来源期刊
BioTechniques
BioTechniques 工程技术-生化研究方法
CiteScore
4.40
自引率
0.00%
发文量
68
审稿时长
3.3 months
期刊介绍: BioTechniques is a peer-reviewed, open-access journal dedicated to publishing original laboratory methods, related technical and software tools, and methods-oriented review articles that are of broad interest to professional life scientists, as well as to scientists from other disciplines (e.g., chemistry, physics, computer science, plant and agricultural science and climate science) interested in life science applications for their technologies. Since 1983, BioTechniques has been a leading peer-reviewed journal for methods-related research. The journal considers: Reports describing innovative new methods, platforms and software, substantive modifications to existing methods, or innovative applications of existing methods, techniques & tools to new models or scientific questions Descriptions of technical tools that facilitate the design or performance of experiments or data analysis, such as software and simple laboratory devices Surveys of technical approaches related to broad fields of research Reviews discussing advancements in techniques and methods related to broad fields of research Letters to the Editor and Expert Opinions highlighting interesting observations or cautionary tales concerning experimental design, methodology or analysis.
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