Using tRNA-linked molecular beacons to image cytoplasmic mRNAs in live cells
Using tRNA-linked molecular beacons to image cytoplasmic mRNAs in live cells
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DOI：
10.1038/nprot.2006.242
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Tyagi, Sanjay
中科院分区：
文献类型：
--
作者：
Mhlanga, Musa M.;Tyagi, Sanjay
活细胞中基因表达的成像产品将为细胞生物学提供独特的见解。分子信标可以通过打开猝灭的荧光团的荧光来报告RNA靶标的存在，因此可以成为在活细胞中进行mRNA成像的有吸引力的探针。然而，当寡核苷酸探针被引入细胞中时，它们会迅速隔离在细胞核中，使细胞质mRNAs的检测变得困难。我们已经证明，如果一个分子信标与tRNA相连，它就会留在细胞质中，并允许检测到细胞质的mRNAs。在这里，我们描述了两种将分子信标连接到tRNA的方法，并展示了联合分子如何用于对活培养细胞中通常存在于细胞质中的mRNA进行成像。此方案总共需要4天时间才能完成。
Imaging products of gene expression in live cells will provide unique insights into the biology of cells. Molecular beacons make attractive probes for imaging mRNA in live cells as they can report the presence of an RNA target by turning on the fluorescence of a quenched fluorophore. However, when oligonucleotide probes are introduced into cells, they are rapidly sequestered in the nucleus, making the detection of cytoplasmic mRNAs difficult. We have shown that if a molecular beacon is linked to a tRNA, it stays in the cytoplasm and permits detection of cytoplasmic mRNAs. Here we describe two methods of linking molecular beacons to tRNA and show how the joint molecules can be used for imaging an mRNA that is normally present in the cytoplasm in live cultured cells. This protocol should take a total of 4 d to complete.