Direct detection of mRNA expression in microbial cells by fluorescence in situ hybridization using RNase H-assisted rolling circle amplification
Direct detection of mRNA expression in microbial cells by fluorescence in situ hybridization using RNase H-assisted rolling circle amplification
复制标题
使用RNase H辅助滚环扩增通过荧光原位杂交直接检测微生物细胞中的mRNA表达
DOI：
10.1038/s41598-020-65864-7
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发表时间：
2020
影响因子：
4.6
通讯作者：
Okamura Yoshiko
中科院分区：
文献类型：
--
作者：
Takahashi Hirokazu;Horio Kyohei;Kato Setsu;Kobori Toshiro;Watanabe Kenshi;Aki Tsunehiro;Nakashimada Yutaka;Okamura Yoshiko
使用新一代测序的荟萃分析是研究微生物群的有力策略；然而，它不能阐明微生物群中单个微生物的作用。了解哪个细胞表达什么基因对于阐明单个细胞在微生物群中的功能是很重要的。在本报告中，我们开发了一种新的荧光原位杂交（FISH）方法，使用rnase - h辅助滚动环扩增来观察微生物细胞中无反转录的mRNA。我们的研究结果表明，该方法适用于革兰氏阴性和革兰氏阳性微生物，没有任何DNA噪声，并且可以在单细胞水平上直接可视化目标mRNA的表达。因此，当我们的程序与荟萃分析的数据相结合时，可以帮助理解微生物群中单个微生物的作用。
Meta-analyses using next generation sequencing is a powerful strategy for studying microbiota; however, it cannot clarify the role of individual microbes within microbiota. To know which cell expresses what gene is important for elucidation of the individual cell’s function in microbiota. In this report, we developed novel fluorescencein situhybridization (FISH) procedure using RNase-H-assisted rolling circle amplification to visualize mRNA of interest in microbial cells without reverse transcription. Our results show that this method is applicable to both Gram-negative and Gram-positive microbes without any noise from DNA, and it is possible to visualize the target mRNA expression directly at the single-cell level. Therefore, our procedure, when combined with data of meta-analyses, can help to understand the role of individual microbes in the microbiota.