Department of Molecular, Cell, and Systems Biology, University of California, Riverside, California 92521, USA.
RNA. 2020 Feb;26(2):218-227. doi: 10.1261/rna.071605.119. Epub 2019 Nov 21.
High-throughput sequencing has become a standard tool for analyzing RNA and DNA. This method usually needs a cDNA/DNA library ligated with specific 5' and 3' linkers. Unlike mRNA, small RNA often contains modifications including 5' cap or triphosphate and 2'--methyl, requiring additional processing steps before linker additions during cloning processes; due to low expression levels, it is difficult to clone small RNA with a small amount of total RNA. Here we present a new strategy to clone 5' modified or unmodified small RNA in an all-liquid-based reaction carried out in a single PCR tube with as little as 20 ng total RNA. The 7-h cloning process only needs ∼1 h of labor. Moreover, this method can also clone mRNA, simplifying the need to prepare two cloning systems for small RNA and mRNA; the barcoded PCR primers are also compatible with non-cDNA cloning applications, including the preparation of genomic libraries. Not only is our method more convenient for cloning modified RNA than available methods, but it is also more sensitive, versatile, and cost-effective. Moreover, the all-liquid-based reaction can be performed in an automated manner.
高通量测序已成为分析 RNA 和 DNA 的标准工具。这种方法通常需要将 cDNA/DNA 文库与特定的 5' 和 3' 接头连接。与 mRNA 不同，小 RNA 通常包含修饰，包括 5' 帽或三磷酸和 2'--甲基，在克隆过程中添加接头之前需要进行额外的处理步骤；由于表达水平低，很难用少量总 RNA 克隆小 RNA。在这里，我们提出了一种新的策略，即在单个 PCR 管中进行全液相反应，用低至 20 ng 的总 RNA 克隆 5' 修饰或未修饰的小 RNA。7 小时的克隆过程仅需要约 1 小时的工作量。此外，这种方法还可以克隆 mRNA，简化了为小 RNA 和 mRNA 准备两种克隆系统的需求；带条形码的 PCR 引物也与非 cDNA 克隆应用兼容，包括基因组文库的制备。与现有方法相比，我们的方法不仅更方便用于克隆修饰 RNA，而且更灵敏、多功能且具有成本效益。此外，全液相反应可以以自动化方式进行。
