Telomerase Activity Measurement in Magnetically Captured Epithelial Cells: Comparison of Slab-Gel and Capillary Electrophoresis

Published

June 1, 2004

Author(s)

J L. Hess, Donald H. Atha, J Xu, W E. Highsmith

Abstract

We have compared telomerase activity measurements by slab-gel and capillary electrophoresis in human cultured cells and in epithelial cells isolated from clinical blood specimens. The cultured cells were A549 and H125 human cancer cell lines, and the epithelial cells were isolated from the peripheral blood of patients with lung and esophageal cancer. Telomerase activity was determined using the telomerase repeat amplification protocol (TRAP) assay with phosphoimager scanning of slab-gels and by laser-induced fluorescence capillary electrophoresis (LIF-CE). Experiments using A549 and H125 cells were performed to determine the reproducibility of each method and to identify the contribution of each stage of the TRAP/PCR assay to the variability. In these experiments it was found that more than half of the variability (15% of the overall variability of 35%) of the slab-gel method and almost all of the variability (15% of the overall variability of 20%) of the CE method was due to the PCR stage of the TRAP assay. In the clinical samples, classification as positive or negative was by visual inspection of the slab-gel and CE electropherograms for the presence of the characteristic 6 base-pair TRAP ladder and by GeneScan analysis of the CE. We examined several criteria including the use of 3, 4, or 5 TRAP bands as the definition of a positive test. Using the slab-gel method, the 5-band criterion gave 40% sensitivity with 100 % specificity (no false positives in inactive controls).

Citation

Electrophoresis

Volume

25

Issue

12

Pub Type

Journals

Keywords

capillary electrophoresis, telomerase

Citation

Hess, J. , Atha, D. , Xu, J. and Highsmith, W. (2004), Telomerase Activity Measurement in Magnetically Captured Epithelial Cells: Comparison of Slab-Gel and Capillary Electrophoresis, Electrophoresis (Accessed April 19, 2024)