A whole-mount in situ hybridization method for microRNA detection in Caenorhabditis elegans.
A whole-mount in situ hybridization method for microRNA detection in Caenorhabditis elegans.
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DOI：
10.1261/rna.054239.115
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发表时间：
2016-07
期刊：
影响因子：
--
通讯作者：
Kohara Y
中科院分区：
文献类型：
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作者：
Andachi Y;Kohara Y
整体原位杂交（Whole-mount in situ hybridization，WISH）是研究microRNA（miRNAs）时空表达模式的重要方法，为阐明其功能提供了重要线索。第一个用于miRNA检测的WISH方法是在斑马鱼中开发的。虽然这种方法很快适用于其他脊椎动物和果蝇，但WISH分析尚未成功用于检测秀丽隐杆线虫中的miRNA。在这里，我们展示了一种新的WISH方法用于检测C.优雅使用这种方法，在体壁肌肉中检测到mir-1 miRNA，其中mir-1 miRNA的表达和作用先前已经阐明。将该方法应用于let-7家族miRNAs，let-7，mir-48，mir-84和mir-241，揭示了它们不同但部分重叠的表达模式，表明可检测到共享短共同序列的miRNAs。在耗尽成熟miRNA的pash-1突变体中，mir-48 miRNA的信号大大降低，表明通过该方法检测到成熟miRNA。这些结果证明了WISH检测C.优雅
Whole-mount in situ hybridization (WISH) is an outstanding method to decipher the spatiotemporal expression patterns of microRNAs (miRNAs) and provides important clues for elucidating their functions. The first WISH method for miRNA detection was developed in zebrafish. Although this method was quickly adapted for other vertebrates and fruit flies, WISH analysis has not been successfully used to detect miRNAs in Caenorhabditis elegans. Here, we show a novel WISH method for miRNA detection in C. elegans. Using this method, mir-1 miRNA was detected in the body-wall muscle where the expression and roles of mir-1 miRNA have been previously elucidated. Application of the method to let-7 family miRNAs, let-7, mir-48, mir-84, and mir-241, revealed their distinct but partially overlapping expression patterns, indicating that miRNAs sharing a short common sequence were distinguishably detected. In pash-1 mutants that were depleted of mature miRNAs, signals of mir-48 miRNA were greatly reduced, suggesting that mature miRNAs were detected by the method. These results demonstrate the validity of WISH to detect mature miRNAs in C. elegans.