Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells
Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells
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DOI：
10.1101/gr.7179508
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发表时间：
2008-04-01
期刊：
影响因子：
7
通讯作者：
Marra, Marco A.
中科院分区：
文献类型：
--
作者：
Morin, Ryan D.;O'Connor, Michael D.;Marra, Marco A.
微小RNA（miRNAs）作为生物过程的重要调节因子正在崭露头角，尽管其特性尚未得到充分阐明。进一步阐明它们作用的关键在于生成更完整的其在不同细胞状态下的数量及表达变化清单。在此，我们报道一种利用Illumina测序技术检测包括微小RNA在内的小RNA表达的新方法。我们还提出了一套用于注释源自已知微小RNA的序列、识别成熟微小RNA序列的变异性以及识别属于先前未鉴定的微小RNA基因的序列的方法。将这种方法应用于人类胚胎干细胞分化为拟胚体前后的RNA，揭示了334个已知以及104个新的微小RNA基因的序列和表达水平。171个已知的和23个新的微小RNA序列在这两种发育状态之间表现出显著的表达差异。由于序列读取数量的增加，这些文库代表了迄今为止最深层次的微小RNA取样，涵盖了近六个数量级的表达。在任一样本中富集的那些微小RNA的预测靶标具有共同特征。在排名靠前的预测基因靶标中包括那些与分化、细胞周期控制、程序性细胞死亡和转录调控有关的基因。
MicroRNAs (miRNAs) are emerging as important, albeit poorly characterized, regulators of biological processes. Key to further elucidation of their roles is the generation of more complete lists of their numbers and expression changes in different cell states. Here, we report a new method for surveying the expression of small RNAs, including microRNAs, using Illumina sequencing technology. We also present a set of methods for annotating sequences deriving from known miRNAs, identifying variability in mature miRNA sequences, and identifying sequences belonging to previously unidentified miRNA genes. Application of this approach to RNA from human embryonic stem cells obtained before and after their differentiation into embryoid bodies revealed the sequences and expression levels of 334 known plus 104 novel miRNA genes. One hundred seventy-one known and 23 novel microRNA sequences exhibited significant expression differences between these two developmental states. Owing to the increased number of sequence reads, these libraries represent the deepest miRNA sampling to date, spanning nearly six orders of magnitude of expression. The predicted targets of those miRNAs enriched in either sample shared common features. Included among the high-ranked predicted gene targets are those implicated in differentiation, cell cycle control, programmed cell death, and transcriptional regulation.