SYBR Green real-time telomeric repeat amplification protocol for the rapid quantification of telomerase activity

Nucleic Acids Res. 2003 Jan 15;31(2):E3-3. doi: 10.1093/nar/gng003.

Abstract

The sensitive telomeric repeat amplification protocol (TRAP) permits telomerase detection in mammalian cell and tissue extracts with very low telomerase activity levels. Unfortunately, conventional TRAP assays require complex post-amplification procedures, such as polyacrylamide gel electrophoresis and densitometry, to measure telomerase products. Therefore, a real-time quantitative TRAP assay (RQ-TRAP) was optimized in the present study and evaluated in comparison with a commercially available quantitative TRAP kit and by monitoring telomerase activity in human hepatocyte cultures, human hepatoma cell lines and telomerase reconstitution experiments. The novel real-time telomerase detection method has many advantages. Other than sample extraction and real-time cycling, no additional time-consuming steps have to be performed for telomerase quantification; reliable and linear telomerase quantification is possible down to single-cell dilutions without the interference of primer-dimer artifacts, and the costs are less. Moreover, the precision is similar to other amplification-based telomerase quantification assays and the results are comparable to data obtained with two commercially available assays. The closed-tube system reduces the risk of carryover contamination and supports high throughput. In conclusion, RQ-TRAP provides a new tool for the rapid and reliable quantification of telomerase activity.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Cells, Cultured
  • Enzyme-Linked Immunosorbent Assay / methods
  • Enzyme-Linked Immunosorbent Assay / standards
  • Fluorescent Dyes
  • Hepatocytes / enzymology
  • Humans
  • Organic Chemicals*
  • Polymerase Chain Reaction / methods*
  • Polymerase Chain Reaction / standards
  • Repetitive Sequences, Nucleic Acid / genetics*
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Telomerase / genetics
  • Telomerase / metabolism*
  • Telomere / genetics*
  • Tumor Cells, Cultured / enzymology

Substances

  • Fluorescent Dyes
  • Organic Chemicals
  • SYBR Green I
  • Telomerase