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.
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.