利用与转运RNA相连的分子信标对活细胞中的细胞质信使核糖核酸进行成像。
Using tRNA-linked molecular beacons to image cytoplasmic mRNAs in live cells.
作者信息
Mhlanga Musa M, Tyagi Sanjay
机构信息
Public Health Research Institute, 225 Warren Street, Newark, New Jersey 07103, USA.
出版信息
Nat Protoc. 2006;1(3):1392-8. doi: 10.1038/nprot.2006.242.
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.
活细胞中基因表达的成像产物将为细胞生物学提供独特的见解。分子信标是用于活细胞中mRNA成像的有吸引力的探针，因为它们可以通过开启淬灭荧光团的荧光来报告RNA靶标的存在。然而，当将寡核苷酸探针引入细胞时，它们会迅速被隔离在细胞核中，使得检测细胞质mRNA变得困难。我们已经表明，如果将分子信标与tRNA连接，它会留在细胞质中并允许检测细胞质mRNA。在这里，我们描述了将分子信标与tRNA连接的两种方法，并展示了如何将联合分子用于对活培养细胞中通常存在于细胞质中的mRNA进行成像。该方案总共需要4天完成。
Zotero 插件