关键词: 端粒, 基因扩增, 酶联免疫吸附测定, 端粒酶活性, 端粒重复序列扩增, TRAP-ELISA

Abstract:

[Abstract] Objective   To establish methodology to detect telomerase activity based on real-time quantitative PCR
technique combined with telomeric repeat amplification protocol (TRAP). Methods   RQ-TRAP system was developed by combining real- time quantitative PCR technique with conventional TRAP method. Telomerase activity was assessed and compared by RQ-TRAP assay and TRAP connected with enzyme-linked immunosorbent assay (TRAP-ELISA) respectively in12kinds of cells. Results   The RQ-TRAP method was both accurate and specified in measuring telomerase activity in a series dilution of protein extracts from293T cells. The sensitivity of this method was8cells and the amplification efficiency was98.92%. Telomerase activity was not detected in negative control group. Statistical analysis revealed a strong correlation between the two assays (r²=0.7625). Conclusion   The feasibility of RQ-TRAP was proved in this article. Compared with TRAP-ELISA, RQ-TRAP has many advantages. Apart from sample extraction and real-time PCR cycling, no other extra time-consuming steps are needed for telomerase quantification；RQ-TRAP is less costly and more rapid and reliable than TRAP-ELISA for quantification of telomerase activity and it also support high throughput.

Key words: Telomere, gene amplification, enzyme-linked immunosorbent assay, Telomerase activity, Telomeric repeat amplification protocol, TRAP-ELISA