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Development of a highly sensitive RT-PCR luminometric hybridization assay for human telomerase reverse-transcriptase
Katerina Angelopoulou1, Tonia Spiropoulou, Aliki Stathopoulou
1Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Athens, 15772, Greece.
Background:
Telomerase is a general diagnostic and prognostic molecular tumor marker since it is expressed in the majority of human tumors in contrast to most healthy tissues. Alternate splicing of human telomerase reverse transcriptase (hTERT) has been shown to affect telomerase activity.
Patients And Methods:
We have developed a hybridization assay that selectively detects the hTERT beta-plus transcript. Biotinylated PCR products were captured on streptavidin-coated microtiter wells, hybridized with digoxigenin-labeled probes and detected by a highly sensitive luminometric reaction.
Results:
The method was applied in ten colorectal tumor forceps biopsies and their corresponding normal tissues. Six out of ten tumors were positive for hTERT beta plus transcript whereas none of the corresponding normal tissues were found positive. There was a complete concordance between the hybridization assay and real-time PCR. When the method was applied in peripheral blood of 20 breast cancer patients with metastatic disease and 21 healthy blood donors, 14 patients (70%) were found positive while all 21 healthy blood donors were negative.
Conclusion:
The developed hybridization assay is highly sensitive and specific for the detection of hTERT beta-plus transcript in clinical samples.