利用cDNA文库测序鉴定微小RNA和其他小调控RNA。
Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing.
作者信息
Hafner Markus, Landgraf Pablo, Ludwig Janos, Rice Amanda, Ojo Tolulope, Lin Carolina, Holoch Daniel, Lim Cindy, Tuschl Thomas
机构信息
Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, 1230 York Avenue, Box 186, New York, NY 10065, USA.
出版信息
Methods. 2008 Jan;44(1):3-12. doi: 10.1016/j.ymeth.2007.09.009.
Abstract
Distinct classes of small RNAs, 20-32 nucleotides long, play important regulatory roles for diverse cellular processes. It is therefore important to identify and quantify small RNAs as a function of development, tissue and cell type, in normal and disease states. Here we describe methods to prepare cDNA libraries from pools of small RNAs isolated from organisms, tissues or cells. These methods enable the identification of new members or new classes of small RNAs, and they are also suitable to obtain miRNA expression profiles based on clone count frequencies. This protocol includes the use of new deep sequencing methods (454/Roche and Solexa) to facilitate the characterization of diverse sequence pools of small RNAs.
摘要
长度为20 - 32个核苷酸的不同种类小RNA在多种细胞过程中发挥着重要的调控作用。因此，在正常和疾病状态下，根据发育阶段、组织和细胞类型来鉴定和定量小RNA非常重要。在这里，我们描述了从生物体、组织或细胞中分离得到的小RNA池制备cDNA文库的方法。这些方法能够鉴定新的小RNA成员或新的小RNA类别，也适用于基于克隆计数频率获得miRNA表达谱。本方案包括使用新的深度测序方法（454/罗氏和 Solexa）来促进对各种小RNA序列库的表征。
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