一种改进的小非编码RNA cDNA文库构建方法

An improved method of sncRNA cDNA library construction

本文使用RNA Antisense Purification(RAP)技术富集纯化sncRNA,有效去除了5S、5.8SrRNA.联合使用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.在sncRNA3′-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.改进后的sncRNA cDNA文库容量为3×105cfu,重组率为95%,且测序结果显示,获得的cDNA序列具有较好的完整性.使用这种成本低且行之有效的改进方法,为深入研究sncRNA奠定了基础.

5S and 5.8S rRNA were removed efficiently by RNA Antisense Purification （RAP）. More 5r- 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. To avoid using the adenylated adapter, a poly（A） structure was added to sncRNA 3r end and then re- verse transcription was carried out with oligo（dT）16VN-adapter primers. This improved sncRNA cDNA library contained 3- 105 independent clones and the recombination rate was 95%. The sequencing results showed that the sncRNA cDNA sequences had good integrity by employing this improved and cost-effective method, which laid the foundation for the further study of sncRNA.