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SYBR Green实时定量端粒重复序列扩增法检测端粒酶活性
  • ISSN号:0253-9896
  • 期刊名称:《天津医药》
  • 时间:0
  • 分类:R34[医药卫生—基础医学]
  • 作者机构:[1] 石河子大学医学院,832000, [2] 杭州师范大学医学院, [3] 石河子大学生命科学学院
  • 相关基金:国家重大科学研究计划项目(2012CB911200)
中文摘要:

目的:采用实时定量端粒重复序列扩增法(RQ-TRAP法)检测不同细胞端粒酶活性。方法用RQ-TRAP和TRAP-ELISA两种方法同时检测12种细胞的端粒酶活性,并比较两种方法的检测结果。结果 RQ-TRAP方法能准确特异地检测系列稀释的293T细胞蛋白提取液的端粒酶活性,灵敏度可达8个细胞,扩增效率为98.92%。阴性对照组则未检测到端粒酶活性。RQ-TRAP方法测得12个细胞系中端粒酶的活性与TRAP-ELISA方法结果有较强相关性(r2=0.7625)。两种方法检测肿瘤细胞端粒酶活性均高于正常细胞。结论 RQ-TRAP方法检测端粒酶可行,比TRAP-ELISA方法成本低、时间短,且支持高通量,是一种新的可快速可靠定量端粒酶活性的方法。

英文摘要:

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.

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期刊信息
  • 《天津医药》
  • 中国科技核心期刊
  • 主管单位:天津市卫生和计划生育委员会
  • 主办单位:天津市医学科学技术信息研究所
  • 主编:王贺胜
  • 地址:天津市和平区贵州路96号D座
  • 邮编:300070
  • 邮箱:tjyybjb@sina.com
  • 电话:022-23337519
  • 国际标准刊号:ISSN:0253-9896
  • 国内统一刊号:ISSN:12-1116/R
  • 邮发代号:6-3
  • 获奖情况:
  • 两年荣获天津市一级期刊、优秀期刊奖,首届、第四、五、六届华北地区优秀期刊奖,第二、三届华北地区十佳期刊奖,第一、二届北方优秀期刊奖,国家科技期刊方阵双百期刊,全国优秀期刊奖
  • 国内外数据库收录:
  • 俄罗斯文摘杂志,美国化学文摘(网络版),英国农业与生物科学研究中心文摘,波兰哥白尼索引,日本日本科学技术振兴机构数据库,中国中国科技核心期刊,中国北大核心期刊(2004版),中国北大核心期刊(2008版),中国北大核心期刊(2011版),中国北大核心期刊(2000版)
  • 被引量:21279