A highly useful tool for studying lncRNAs is simultaneous RNA–DNA FISH, which reveals the localization and quantitative information of RNA and DNA in cellular contexts. However, a simple combination of RNA FISH and DNA FISH often generates disappointing results because the fragile RNA signals are often damaged by the harsh conditions used in DNA FISH for denaturing the DNA. Here, we describe a robust and simple RNA–DNA FISH protocol, in which amino-labeled nucleic acid probes are used for RNA FISH. The method is suitable to detect single-RNA molecules simultaneously with DNA.
Fluorescence in situ hybridization (FISH) Single-molecule RNA FISH Amino-labeled probes Simultaneous RNA–DNA FISH lncRNA
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L-FZ is supported by the Singapore National Research Foundation under its Cooperative Basic Research Grant administered by the Singapore Ministry of Health’s National Medical Research Council. FS is supported by Nanyang Assistant Professor Fellowship (M4080531) and Ministry of Education Tier 2 Research Grant (M4020163). The authors would also like to acknowledge the Nanyang Technological University College of Science Collaborative Research Award.