一种改进的小非编码RNA cDNA文库构建方法
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Q78

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国家自然科学基金项目 (31371325)


An improved method of sncRNA cDNA library construction
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    摘要:

    小非编码RNA(small noncoding RNA, sncRNA)是一类长度小于200个核苷酸且缺乏蛋白质编码能力的RNA. 随着对miRNA、piRNA等sncRNA的深入研究,人们逐渐认识到sncRNA在基因表达调控、可变剪接、细胞生长、分化、增殖、凋亡与疾病等方面发挥着重要作用. 构建sncRNA cDNA文库是获取和研究sncRNA的主要途径. 目前, sncRNA cDNA文库构建试剂盒价格昂贵,因此,本研究对sncRNA cDNA文库构建方法进行改进: (1)使用RNA Antisense Purification (RAP)技术富集纯化sncRNA,有效去除了5S、5.8S rRNA. (2)使用Tobacco Acid Pyrophosphatase(TAP) 和RNA 5′ Polyphosphatase处理sncRNA,将5′-端为非单磷酸结构的sncRNA转变成单磷酸结构,增加了文库覆盖率. (3)在sncRNA 3′-端添加poly(A)尾结构以及使用oligo(dT)16VN-adapter锚定引物进行逆转录,间接解决了sncRNA 3′-端接头定向连接问题,不再使用价格昂贵的腺苷化接头. 使用这种成本低且行之有效的改进方法,最终可以得到完整的sncRNA cDNA序列,为深入研究sncRNA奠定了基础.

    Abstract:

    With the increasing recognization of sncRNA, especially miRNA and piRNA, it has been proved that small noncoding RNA (sncRNA,<200 nt) plays a significant role at many levels including gene expression regulation, alternative splicing, cell growth, differentiation, proliferation, apoptosis and disease, etc. The sncRNA cDNA library construction is the main way to acquire and research sncRNA. Now, there are many sncRNA cDNA library kits on the market but it’s too expensive. Therefore, an improved method of sncRNA cDNA library construction was established: (1)5S and 5.8S rRNA were removed efficiently by RNA Antisense Purification (RAP); (2) More 5′-monophosphorylated RNAs were acquired after using Tobacco Acid Pyrophosphatase (TAP) and RNA 5' sncRNA Polyphosphatase, which resulted in the increasement of the coverage of the cDNA library. (3) To avoid using the adenylated adapter, a poly(A) structure was added to sncRNA 3′ end and then reverse transcription was carried out with oligo(dT)16VN-adapter primers. Finally, the complete sncRNA cDNA sequences were obtained by employing this improved and cost-effective method, which laid the foundation for the further study of sncRNA.

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引用本文格式: 杜光石,罗发涛,肖燕,陈红军,吴传芳. 一种改进的小非编码RNA cDNA文库构建方法[J]. 四川大学学报: 自然科学版, 2017, 54: 653.

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  • 收稿日期:2016-03-15
  • 最后修改日期:2016-05-26
  • 录用日期:2016-05-26
  • 在线发布日期: 2017-06-03
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