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Poly(A)加尾-RNase H消化-RT-PCR法分析应激诱导生成5'端tRNA半分子 被引量：1

Detection of stress-induced 5' tRNA halves by poly(A) tailed-RNase H digestion-RTPCR
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摘要 目的建立一种简便、快速检测应激诱导细胞生成的5'端转移RNA(tRNA)半分子的方法。方法提取应激诱导细胞RNA,用poly(A)加尾酶在RNA分子3'端加尾后,加入与待测tRNA的3'端部分序列互补的简并DNA探针杂交,经RNase H消化与DNA探针互补的tRNA序列,再由oligo(d T)n锚定引物进行逆转录获得互补DNA(c DNA),以5'端tRNA半分子和锚定引物的序列为PCR反应的上下游引物,PCR扩增检测5'端tRNA半分子。结果经poly(A)加尾-RNase H消化-RT-PCR-电泳等步骤,可以实现对5'端tRNA半分子的检测分析。结论 poly(A)加尾-RNase H消化-RT-PCR法的建立实现了5'端tRNA半分子的简便、快速检测分析,为tRNA半分子生物学功能研究和检测分析提供了工具。 Objective To develop a simple and quick method for detection of stress-induced 5＇ transfer RNA（ tRNA）halves. Methods Total RNA purified from stress induced cells was polyadenylated by poly（ A） polymerase,and then degenerate DNA probes were used to hybridize with 3＇ tRNA-halves of intact tRNAs,while RNase H specifically degraded the 3＇tRNA-halves strand in tRNA-DNA probes hybrids. Using the RNase H digestion total RNA as templates,complementary DNA（ c DNA） was synthesized by oligo（ dT） n-anchored primers. The primer of 5＇ tRNA halves and anchored-primer were used to amplify 5＇ tRNA halves by PCR. Results The results showed that the method of poly（ A）-tailed-RNase H digestion- RT-PCR could be successfully used to detect stress-induced 5＇ tRNA halves. Conclusion A simple and quick method for detection of 5＇ tRNA halves has been established,which is a user-friendly tool for 5＇ tRNA halves detection and function research.
作者 刘荻 付汉江 刘继来 朱捷 郑晓飞
机构地区 安徽医科大学研究生学院 军事医学科学院放射与辐射医学研究所
出处 《军事医学》 CAS CSCD 北大核心 2015年第6期460-463,共4页 Military Medical Sciences
基金 国家自然科学基金资助项目(31070702 31270836) 国家高技术研究发展计划资助项目(2012AA022501)
关键词 RNA 转移 tRNA半分子 poly(A)加尾酶 逆转录聚合酶链反应 内切核酸酶类 RNASE H 分析 tRNA 5＇ tRNA half poly（A） polymerase reverse transcriptase polymerase chain reaction endonucleases RNase H detection
分类号 Q522 [生物学—生物化学] Q503 [生物学—生物化学]
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参考文献14

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同被引文献28

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  • 5Thompson DM, Parker R. Stressing out over tRNA cleavage[J]. Cell,2009,138(2):215-219.
  • 6Wilusz JE,Freier SM,Spector DL.3' end processing of a long nuclear-retained noncoding RNA yields a tRNA-like cytoplasmic RNA [J]. Cell,2008,135(5):919-932.
  • 7Yamasaki S, Ivanov P, Hu GF, et al. Angiogenin cleaves tRNA and promotes stress-induced translational repression[J]. J Cell Biol,2009,185(1):35-42.
  • 8Lee SR,Collins K.Starvation-induced cleavage of the tRNA anticodon loop in tetrahymena thermophila[J].J Biol Chem,2005,280(52):42744-42749.
  • 9Fu H, Feng J, Liu Q, et al. Stress induces tRNA cleavage by angiogenin in mammalian cells[J]. FEBS Lett,2009,583(2):437-442.
  • 10Lee YS, Shibata Y, Malhotra A, et al. A novel class of small RNAs: tRNA-derived RNA fragments (tRFs) [J]. Genes Dev,2009,23(22):2639-2649.

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军事医学
2015年 第6期

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