Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, USA.
Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, TX, USA.
Methods Mol Biol. 2021;2200:225-254. doi: 10.1007/978-1-0716-0880-7_11.
RNA silencing plays a critical role in diverse biological processes in plants including growth, development, and responses to abiotic and biotic stresses. RNA silencing is guided by small non-coding RNAs (sRNAs) with the length of 21-24 nucleotides (nt) that are loaded into Argonaute (AGO) to repress expression of target loci and transcripts through transcriptional or posttranscriptional gene silencing mechanisms. Identification and quantitative characterization of sRNAs are crucial steps toward appreciation of their functions in biology. Here, we developed a step-by-step protocol to precisely illustrate the process of cloning of sRNA libraries and correspondingly computational analysis of the recovered sRNAs. This protocol can be used in all kinds of organisms, including Arabidopsis, and is compatible with various high-throughput sequence technologies such as Illumina Hiseq. Thus, we wish that this protocol represents an accurate way to identify and quantify sRNAs in vivo.
RNA 沉默在植物的多种生物学过程中发挥着关键作用，包括生长、发育以及对非生物和生物胁迫的响应。RNA 沉默由长度为 21-24 个核苷酸 (nt) 的小非编码 RNA (sRNA) 指导，这些 sRNA 被装载到 Argonaute (AGO) 中，通过转录或转录后基因沉默机制来抑制靶基因座和转录物的表达。sRNA 的鉴定和定量特征分析是了解其在生物学中功能的关键步骤。在这里，我们开发了一个逐步的方案，精确地说明了 sRNA 文库克隆的过程以及相应的回收 sRNA 的计算分析。该方案可用于包括拟南芥在内的各种生物体，并且与各种高通量测序技术（如 Illumina Hiseq）兼容。因此，我们希望该方案代表一种在体内鉴定和定量 sRNA 的准确方法。
