Construction of small RNA cDNA libraries for deep sequencing.
Construction of small RNA cDNA libraries for deep sequencing.
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DOI：
10.1007/978-1-60761-811-9_7
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发表时间：
2010
影响因子：
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通讯作者：
Molly F. Thomas;K. Ansel
中科院分区：
文献类型：
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作者：
Molly F. Thomas;K. Ansel
由于小rna介导的基因沉默现象在15年前首次被描述（Lee等人）。Cell 75:843-854, 1993；Wightman等人。Cell 75:855-862, 1993)，很明显，多种内源性小rna在建立和维持细胞系中起着重要作用。特别是MicroRNAs (miRNAs)，已被证明对构成哺乳动物免疫系统的许多特化细胞的发育和功能施加调节控制（Baltimore等）。中华医学杂志，2009；中国生物医学工程学报（英文版），2008；Xiao and Rajewsky Cell, 136:26-36, 2009)。下一代测序仪的出现为分析许多不同细胞类型的小RNA转录组提供了重要的工具。与传统的桑格测序相比，下一代测序机可以并行处理数百万个序列读取，在短短几天内生成兆级数据。用于测序的小RNA文库的生成相对简单，包括将平台特异性适配器序列连接到小RNA上，然后对连接的物种进行逆转录和PCR扩增。虽然其他基于杂交的技术可用于分析具有良好特征的小rna，但高通量测序仍然是发现新型小rna和转录后编辑的最强大方法。
Since the phenomenon of small RNA-mediated gene silencing was first described over 15 years ago (Lee et al. Cell 75:843–854, 1993; Wightman et al. Cell 75:855–862, 1993), it has become evident that a variety of endogenous small RNAs play an important role in establishing and maintaining cell lineages. MicroRNAs (miRNAs), in particular, have been shown to exert regulatory control over the development and function of the many specialized cells that comprise the mammalian immune system (Baltimore et al. Nat Immunol 9:839–845, 2008; Kanellopoulous and Monticelli Semin Cancer Biol 18:79–88, 2008; Xiao and Rajewsky Cell 136:26–36, 2009). The advent of next generation sequencers provides an important tool for profiling the small RNA transcriptome of many diverse cell types. Compared to traditional Sanger sequencing, next generation sequencing machines can process millions of sequence reads in parallel, generating megabases of data within just a few days. The generation of small RNA libraries for sequencing is relatively straightforward and involves the ligation of platform-specific adapter sequences to small RNAs, followed by reverse transcription of the ligated species and PCR amplification. While other hybridization-based techniques are available for profiling well-characterized small RNAs, high-throughput sequencing remains the most powerful method for discovering novel small RNAs and posttranscriptional editing.