Development of a highly sensitive RT-PCR luminometric hybridization assay for human telomerase reverse-transcriptase beta-plus transcript.
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.


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.


CONCLUSIONS
The developed hybridization assay is highly sensitive and specific for the detection of hTERT beta-plus transcript in clinical samples.
Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Athens, 15772, Greece.
Katerina
K
Angelopoulou
Tonia
T
Spiropoulou
Aliki
A
Stathopoulou
Dimitris
D
Dimitroulopoulos
Nikos
N
Malamos
Vassilis
V
Georgoulias
Emanouil
E
Paraskevas
Evi S
ES
Lianidou
D017398:Alternative Splicing; D001483:Base Sequence; D014408:Biomarkers, Tumor / Q000235:genetics; D001706:Biopsy; D019921:Biotinylation; D003110:Colonic Neoplasms / Q000201:enzymology / Q000235:genetics / Q000473:pathology / Q000601:surgery; D017931:DNA Primers; D004268:DNA-Binding Proteins; D014644:Genetic Variation; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods; D008163:Luminescent Measurements; D009367:Neoplasm Staging; D012004:Rectal Neoplasms / Q000201:enzymology / Q000235:genetics / Q000473:pathology / Q000601:surgery; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019098:Telomerase / Q000235:genetics*; D014158:Transcription, Genetic
D014408:Biomarkers, Tumor; D017931:DNA Primers; D004268:DNA-Binding Proteins; D019098:Telomerase