Preparation and characterization of candidate reference materials for telomerase assays.
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 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.


RESULTS
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).


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.
Jakupciak
John P
JP
Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
Barker
Peter E
PE
Wang
Wendy
W
Srivastava
Sudhir
S
Atha
Donald H
DH
D014408:Biomarkers, Tumor / Q000032:analysis; D045744:Cell Line, Tumor; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D009369:Neoplasms / Q000175:diagnosis / Q000201:enzymology; D016133:Polymerase Chain Reaction / Q000379:methods; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D012015:Reference Standards; D012091:Repetitive Sequences, Nucleic Acid; D019098:Telomerase / Q000235:genetics / Q000378:metabolism* / Q000592:standards; D016615:Telomere / Q000235:genetics
D014408:Biomarkers, Tumor; D021122:Protein Subunits; D012333:RNA, Messenger; D019098:Telomerase