Imaging individual microRNAs in single mammalian cells in situ.
Imaging individual microRNAs in single mammalian cells in situ.
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DOI：
10.1093/nar/gkp482
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发表时间：
2009-08
影响因子：
14.9
通讯作者：
Tsourkas A
中科院分区：
文献类型：
--
作者：
Lu J;Tsourkas A
MicroRNAs （miRNAs）是基因表达的有效负调控因子，涉及大多数主要的细胞过程。尽管我们对miRNA的生物发生和机制的理解取得了快速进展，但关于miRNA的功能及其对细胞周期控制的影响仍存在许多基本问题。考虑到最近关于细胞间基因表达波动对表型多样性影响的报道，观察细胞群体的平均miRNA表达可能会导致丢失连接miRNA表达和细胞功能的重要信息。然而，目前还没有有效的技术来量化单细胞水平上的miRNA表达。在这里，描述了一种方法，用于检测单个miRNA分子在癌细胞使用荧光原位杂交。该方法将锁定核酸探针的独特识别特性与酶标记荧光相结合。使用这种方法，单个mirna被识别为明亮的、光稳定的荧光点。在本研究中，miR-15a在MDA-MB-231和HeLa细胞中被量化，miR-155在MCF-7细胞中被量化。发现动态范围跨越三个数量级，每个细胞的平均miRNA拷贝数在定量RT-PCR获得的测量值的17.5%以内。
MicroRNAs (miRNAs) are potent negative regulators of gene expression that have been implicated in most major cellular processes. Despite rapid advances in our understanding of miRNA biogenesis and mechanism, many fundamental questions still remain regarding miRNA function and their influence on cell cycle control. Considering recent reports on the impact of cell-to-cell fluctuations in gene expression on phenotypic diversity, it is likely that looking at the average miRNA expression of cell populations could result in the loss of important information connecting miRNA expression and cell function. Currently, however, there are no efficient techniques to quantify miRNA expression at the single-cell level. Here, a method is described for the detection of individual miRNA molecules in cancer cells using fluorescence in situ hybridization. The method combines the unique recognition properties of locked nucleic acid probes with enzyme-labeled fluorescence. Using this approach, individual miRNAs are identified as bright, photostable fluorescent spots. In this study, miR-15a was quantified in MDA-MB-231 and HeLa cells, while miR-155 was quantified in MCF-7 cells. The dynamic range was found to span over three orders of magnitude and the average miRNA copy number per cell was within 17.5% of measurements acquired by quantitative RT-PCR.
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影响因子：
64.8
作者：
Lu, J;Getz, G;Golub, TR
通讯作者：
Golub, TR
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