Preparation and Characterization of Candidate Reference Materials for Telomerase Assays

• Published in: Clinical chemistry (Baltimore, Md.) Md.), 2005-08, Vol.51 (8), p.1443-1450
• Main Authors: Jakupciak, John P, Barker, Peter E, Wang, Wendy, Srivastava, Sudhir, Atha, Donald H
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Biomarkers, Tumor - analysis; Cell Line, Tumor; Electrophoresis, Capillary; Electrophoresis, Polyacrylamide Gel; Freezing; Fundamental and applied biological sciences. Psychology; Humans; Investigative techniques, diagnostic techniques (general aspects); Medical sciences; Neoplasms - diagnosis; Neoplasms - enzymology; Polymerase Chain Reaction - methods; Protein Subunits - genetics; Protein Subunits - metabolism; Reference Standards; Repetitive Sequences, Nucleic Acid; RNA, Messenger - analysis; Telomerase - genetics; Telomerase - metabolism; Telomerase - standards; Telomere - genetics
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2004.044727

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. We prepared a CHAPS buffer lysate from cultured A549 cells and stored it at -80 degrees 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. Preparations made after flash freezing and sonication of cells were approximately 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). 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.