MiR-RACE, a new efficient approach to determine the precise sequences of computationally identified trifoliate orange (Poncirus trifoliata) microRNAs.
MiR-RACE, a new efficient approach to determine the precise sequences of computationally identified trifoliate orange (Poncirus trifoliata) microRNAs.
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DOI：
10.1371/journal.pone.0010861
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发表时间：
2010-06-07
期刊：
影响因子：
3.7
通讯作者：
Ma Z
中科院分区：
文献类型：
--
作者：
Song C;Fang J;Wang C;Guo L;Nicholas KK;Ma Z
在报道的数百种植物中编码miRNAs的基因中，更多的是通过多种计算方法预测的。然而，与由起始密码子和终止密码子定义的蛋白质编码基因不同，miRNA分子的末端并不具有可以用来精确定义成熟miRNA的特征，这使得计算miRNA预测方法往往不能以核苷酸水平的精确度预测成熟miRNA在前体中的准确位置。据我们所知，目前还没有关于确定这些miRNAs的精确序列，特别是两端序列的综合策略的报道。在这项研究中，我们报告了一种有效的方法来确定精确的计算预测的microRNA（miRNAs）的序列，该方法结合了miRNA富集文库制备，两个特定的5′和3′ miRNA RACE（miR-RACE）PCR反应，和序列定向克隆，其中最具挑战性的步骤是两个特定的基因特异性引物设计的两个RACE反应。通过RLM-5′ RACE和测序对miRNA介导的mRNA切割进行验证。实时荧光定量PCR检测各miRNA的表达。使用计算预测的9个枳橙子（Poncirus trifoliata）miRNAs验证了这种新开发的方法的效率。通过miR-RACE和测序验证计算鉴定的miRNA。定量分析表明，它们具有不同的表达。通过检测枳中miRNA介导的mRNA切割，实验验证了8个靶基因。本文开发的有效和强大的方法可以成功地用于验证miRNA的序列，特别是末端，其描绘了计算预测的前体中的完整miRNA序列。
Among the hundreds of genes encoding miRNAs in plants reported, much more were predicted by numerous computational methods. However, unlike protein-coding genes defined by start and stop codons, the ends of miRNA molecules do not have characteristics that can be used to define the mature miRNAs exactly, which made computational miRNA prediction methods often cannot predict the accurate location of the mature miRNA in a precursor with nucleotide-level precision. To our knowledge, there haven't been reports about comprehensive strategies determining the precise sequences, especially two termini, of these miRNAs. In this study, we report an efficient method to determine the precise sequences of computationally predicted microRNAs (miRNAs) that combines miRNA-enriched library preparation, two specific 5′ and 3′ miRNA RACE (miR-RACE) PCR reactions, and sequence-directed cloning, in which the most challenging step is the two specific gene specific primers designed for the two RACE reactions. miRNA-mediated mRNA cleavage by RLM-5′ RACE and sequencing were carried out to validate the miRNAs detected. Real-time PCR was used to analyze the expression of each miRNA. The efficiency of this newly developed method was validated using nine trifoliate orange (Poncirus trifoliata) miRNAs predicted computationally. The miRNAs computationally identified were validated by miR-RACE and sequencing. Quantitative analysis showed that they have variable expression. Eight target genes have been experimentally verified by detection of the miRNA-mediated mRNA cleavage in Poncirus trifoliate. The efficient and powerful approach developed herein can be successfully used to validate the sequences of miRNAs, especially the termini, which depict the complete miRNA sequence in the computationally predicted precursor.
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