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Related Concept Videos

FISH - Fluorescent In-situ Hybridization02:07

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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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Droplet Digital TRAP ddTRAP: Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction
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Optimized for routine: highly sensitive fluorescent Telomeric Repeat Amplification Protocol (f-TRAP).

Silke Fähnrich1, Anne Wedemann1,2, Laura Steenpass1

  • 1Department of Human and Animal Cell Lines, Leibniz-Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany.

Biotechniques
|December 15, 2024
PubMed
Summary

We developed a sensitive assay to measure telomerase activity (TA), crucial for cell division and cancer research. This optimized method detects one telomerase-positive cell among ten thousand, aiding in stem cell and oncology studies.

Keywords:
capillary electrophoresisfluorescent telomeric repeat amplification protocol (f-TRAPhuman telomerase reverse transcriptase (hTERT)immortalizationnon-radioactivetelomerase activity (TA)

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Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay
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Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay
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Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Telomerase activity (TA) is suppressed in differentiated human cells, leading to chromosome shortening and the Hayflick limit.
  • This mechanism acts as a tumor suppression surveillance system.
  • Assessing TA is vital for evaluating cell line immortality and induced pluripotent stem cells (iPSCs).

Purpose of the Study:

  • To present an optimized protocol for measuring telomerase activity.
  • To achieve a highly sensitive detection limit for TA.
  • To provide a robust assay for research and clinical oncology applications.

Main Methods:

  • Combines a fluorescently labeled bait primer with polymerase chain reaction (PCR) amplification.
  • Utilizes analytical capillary electrophoresis (CE) for detection.
  • Optimized for routine use, from single measurements to high-throughput analysis with triplicate lysate concentrations.

Main Results:

  • Achieved a detection limit of one telomerase-positive cell per ten thousand negative cells.
  • The f-TRAP protocol is optimized for speed and routine laboratory application.
  • Standardized data processing and storage are facilitated by CE analysis.

Conclusions:

  • The optimized f-TRAP assay offers a sensitive and efficient method for measuring telomerase activity.
  • This assay can be applied to assess cell immortality, iPSC reprogramming, and has potential in clinical oncology.
  • Standardized data analysis ensures reliable documentation and facilitates wider adoption in research and diagnostics.