Linsen Sam E V, Cuppen Edwin
Hubrecht Institute, Utrecht, The Netherlands.
Methods Mol Biol. 2012;822:205-17. doi: 10.1007/978-1-61779-427-8_14.
Digital gene expression (DGE) profiling techniques are playing an eminent role in the detection, localization, and differential expression quantification of many small RNA species, including microRNAs (1-3). Procedures in small RNA library preparation techniques typically include adapter ligation by RNA ligase, followed by reverse transcription and amplification by PCR. This chapter describes three protocols that were successfully applied to generate small RNA sequencing SOLiD(TM) libraries. The Ambion SREK(TM)-adopted protocol can be readily used for multiplexing samples; the modban-based protocol is cost-efficient, but biased toward certain microRNAs; the poly(A)-based protocol is less biased, but less precise because of the A-tail that is introduced. In summary, each of these protocols has its advantages and disadvantages with respect to the ease of including barcodes, costs, and outcome.
数字基因表达（DGE）谱分析技术在许多小RNA种类（包括微小RNA，1 - 3）的检测、定位和差异表达定量方面发挥着重要作用。小RNA文库制备技术的步骤通常包括通过RNA连接酶进行接头连接，随后进行逆转录和PCR扩增。本章描述了三种成功应用于生成小RNA测序SOLiD™文库的方案。采用Ambion SREK™的方案可轻松用于样本复用；基于modban的方案成本效益高，但对某些微小RNA存在偏向性；基于poly(A)的方案偏向性较小，但由于引入了A尾而不太精确。总之，这些方案在包含条形码的难易程度、成本和结果方面各有优缺点。
