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Fluorescence in situ hybridization, or FISH, was developed in the early 1980s and has quickly become one of the most widely used techniques in cytogenetics. Labeled probes are used to bind complementary DNA or RNA sequences on a chromosome or in a region within a cell. Earlier, the probes could only be obtained by cloning or reverse transcription of a DNA template. Currently, the probe oligonucleotides can be synthesized synthetically. Additionally, with the advancement of optical techniques,...
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优化为常规:高度敏感的光端粒重复放大协议 (f-TRAP).

Silke Fähnrich1, Anne Wedemann1,2, Laura Steenpass1

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概括

我们开发了一种敏感的测试方法来测量端粒酶活性 (TA),这对于细胞分裂和癌症研究至关重要. 这种优化的方法在一万个细胞中检测到一个端粒酶阳性细胞,有助于干细胞和瘤学研究.

关键词:
毛细血管电泳 毛细血管电泳光端粒重复放大协议 (f-TRAP) 的使用.人类端粒酶逆转录酶 (hTERT)永生化就是永生化.非放射性的非放射性物质.端粒酶活动 (TA)

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科学领域:

  • 生物化学 生化学
  • 分子生物学分子生物学
  • 细胞生物学 细胞生物学

背景情况:

  • 端粒酶活性 (TA) 在分化的人类细胞中被抑制,导致染色体缩短和海夫利克极限.
  • 这个机制充当了瘤抑制监控系统.
  • 评估TA对于评估细胞系不朽性和诱导多能干细胞 (iPSCs) 是至关重要的.

研究的目的:

  • 为测量端粒酶活性提供一个优化的协议.
  • 为了实现TA的高度敏感的检测极限.
  • 为研究和临床瘤学应用提供可靠的试验.

主要方法:

  • 结合了光标记的诱原料和聚合酶连锁反应 (PCR) 放大.
  • 使用分析毛细血管电泳 (CE) 进行检测.
  • 优化用于常规使用,从单次测量到高通量分析,用三倍酸盐度进行分析.

主要成果:

  • 在每一万个阴性细胞中达到1个端粒酶阳性细胞的检测极限.
  • 该f-TRAP协议是优化速度和例行实验室应用.
  • 通过CE分析,标准化数据处理和存储变得更加容易.

结论:

  • 优化的f-TRAP测定为测量端粒酶活性提供了一种敏感且高效的方法.
  • 这种测试可用于评估细胞不死亡性,iPSC重编程,并且在临床瘤学中具有潜力.
  • 标准化的数据分析确保了可靠的文档,并促进了在研究和诊断中更广泛的采用.