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 in 12 kinds of cells. Results The RQ-TRAP method was both accurate and specified in measuring telomerase activity in a series dilution of protein extracts from 293T cells. The sensitivity of this method was 8 cells and the amplification efficiency was 98.92%. Telomerase activity was not detected in negative control group. Statistical analysis revealed a strong correlation between the two assays (r2=0.762 5). 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.
ABSTRACT
Purpose:To investigate the telomerase activity in human digestive tract cancer and its significance and evaluate usefulness of the TRAP ELISA method .Methods:Telomerase activity was examined in 112 tumor specimens, including the following cancers: esophageal(12), gastric(36), hepatic(15), pancreatic (11), colorectal(38) and paracancerous tissues(94) by TRAP ELISA method.Results:The telomerase activity in tumor tissues was significantly higher than in paracancerous and normal tissues ( P
ABSTRACT
Objective To investigate the effects of human telomerase reverse transcriptase(hTERT) and c-myc antisense oligodeoxynucleotide(ASODN) on the telomerase activity in HL-60 cells,and to explore the relationship between the telomerase activity and the expression of hTERT and c-myc in HL-60 cells. Methods The expression of hTERT and c-myc was sealed by ASODN in HL-60 cells,and was detected by RT-PCR.The telomerase activity was determined with TRAP-ELISA and PAGE-silver staining. Results After treated with ASODN for 72 hours the expression of hTERT and c-myc mRNA was inhibited in different degree,and was the lowest in group of 30??mol/L ASODN.As for the TRAP-ELISA detection,the difference was significant when compared with groups of 10,20,30??mol/L hTERT ASODN and 20、30??mol/L c-myc ASODN with group of untreated(P0.05).PAGE-silver staining detection showed that comparing ASODN groups with SODN groups the telomerase image strips were decreased and was the least in group of 30??mol/L,and the telomerase image strips were also decreased when compared the same density group of hTERT ASODN with c-myc ASODN.Conclusion hTERT and c-myc ASODN may inhibit the expression of the genes respectively and down-regulate the telomerase activity in HL-60 cells.