Objectives: To develop a sensitive telomeric repeat amplification protocol (TRAP)-silver staining assay for telomerase activity quantification.
Design and methods: TRAP assays were performed by using a TRAPeze telomerase kit with or without [alpha-32P]-dCTP. Amplification products were electrophoresed in polyacrylamide gels and detected by autoradiography or a modified silver staining protocol. Telomerase activity was quantified from radioactive counts or optical density of telomerase products from test extracts and controls.
Results: TRAP-silver staining assay was at least as sensitive as radioactive TRAP assay and quantified telomerase activity within linearity from 10 to 3,000 cell equivalents. Both methods quantified a weak telomerase activity in normal endometrial glandular epithelial cells (GEC) and a strong increase in immortalized GEC. In human pathologic endometria (n=24), telomerase activity was correlated with lesion seriousness and distinguished simple hyperplasias from nonhyperplasic or cancerous lesions.
Conclusions: TRAP-silver staining assay is suitable for cell and tissue telomerase activity routine quantification.