Clinical Chemistry
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Clinical Chemistry 51: 1443-1450, 2005. First published June 10, 2005; 10.1373/clinchem.2004.044727
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(Clinical Chemistry. 2005;51:1443-1450.)
© 2005 American Association for Clinical Chemistry, Inc.

Cancer Diagnostics

Preparation and Characterization of Candidate Reference Materials for Telomerase Assays

John P. Jakupciak1, Peter E. Barker1, Wendy Wang2, Sudhir Srivastava2 and Donald H. Atha1,a

1 Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD.
2 Cancer Biomarkers Research Group, National Cancer Institute, Rockville, MD.

aAddress correspondence to this author at: NIST, Biotechnology Division, 100 Bureau Dr., MS 8311, Gaithersburg, MD 20899. Fax 301-975-8505; e-mail donald.atha{at}nist.gov.

Background: Telomerase has been measured in body fluids of cancer patients, and clinical tests for telomerase may have utility as noninvasive, cost-effective methods for the early detection of cancer. However, telomerase activity measured by common methods such as the telomerase repeat amplification protocol (TRAP) and telomerase reverse transcriptase catalytic subunit (hTERT) mRNA by reverse transcription-PCR (RT-PCR) varies among laboratories.

Methods: We prepared a CHAPS buffer lysate from cultured A549 cells and stored it at –80 °C. Telomerase activity was measured by TRAP/PCR and real-time TRAP/PCR in conjunction with RT-PCR measurements of hTERT mRNA. Activity measured with use of the robot-assisted TRAP (RApidTRAP) multicapillary electrophoresis system was compared with single-capillary and slab-gel measurements in the range 10 to 10 000 cell equivalents.

Results: Preparations made after flash freezing and sonication of cells were ~3-fold more active. Although the slab-gel and capillary instruments detected telomerase activity, the multicapillary instrument was better suited for high-throughput studies. Measurements of telomerase by TRAP/real-time PCR and hTERT mRNA/RT-PCR yielded reproducible titrations in the range 10 to 10 000 cell equivalents (CVs, 1%–8% and 1%–3%, respectively).

Conclusions: We have prepared and characterized a candidate reference material that appears to be suitable for use in a wide range of assays of telomerase activity and expression.






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