Abstract
RNA in situ hybridization reveals the abundance and location of gene expression in cells or tissues, providing a technical basis for the clinical diagnosis of diseases. In this chapter, we show a “V” shape probe-mediated single-molecule chromogenic in situ hybridization (vsmCISH) technique for bright-field visualization of individual RNA molecules. In our method, several pairs of target hybridization probes are hybridized to RNA molecules and each probe pair forms a “V” shape overhang. The overhang oligonucleotides then mediated the proximity ligation to form DNA circles, followed by rolling circle amplification for signal enhancement and enzyme-catalyzed chromogenic reaction-based readout. The colorimetric assay avoids problems such as photobleaching and autofluorescence of current fluorescent in situ hybridization-based single-molecule RNA detection techniques. Furthermore, the relatively straightforward protocol makes the method useful for biological research and clinical diagnosis applications.
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Acknowledgments
This study was supported by the Natural Science Foundation of Fujian Province (2022J06022 and 2021Y4001), the Quanzhou Science and Technology Plan Project (2021C040R), and the Scientific Research Funds of Huaqiao University.
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Xia, X., Jiang, M., Lin, C., Ke, R. (2024). Visualization of Individual RNA Molecules by Proximity Ligation-Based Chromogenic In Situ Hybridization Assay. In: Astatke, M. (eds) RNA Amplification and Analysis. Methods in Molecular Biology, vol 2822. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3918-4_11
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DOI: https://doi.org/10.1007/978-1-0716-3918-4_11
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Publisher Name: Humana, New York, NY
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Online ISBN: 978-1-0716-3918-4
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