Journal
RNA
Volume 25, Issue 8, Pages 1038-1046Publisher
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1261/rna.070599.119
Keywords
chromogenic in situ hybridization; padlock probe; rolling circle amplification; single molecule
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Funding
- National Key Research and Development Program of China [2017YFA0106800]
- National Natural Science Foundation of China [31770927]
- Natural Science Foundation of Fujian Province [2019J01072]
- Subsidized Project for Postgraduate's Innovative Fund in Scientific Research of Huaqiao University
- Scientific Research Funds of Huaqiao University [600005-Z16Y0008]
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Visualization of gene expression at single RNA molecular level represents a great challenge to both imaging technologies and molecular engineering. Here we show a single molecule chromogenic in situ hybridization (smCISH) assay that enables counting and localizing individual RNA molecules in fixed cells and tissue under bright-field microscopy. Our method is based on in situ padlock probe assays directly using RNA as a ligation template and rolling circle amplification combined with enzyme catalyzed chromogenic reaction for amplification product visualization. We show potential applications of our method by detecting gene expression variations in single cells, subcellular localization information of expressed genes, and gene expression heterogeneity in formalin-fixed, paraffin-embedded tissue sections. This facile and straightforward method can in principle be applied to any type of RNA molecules in different samples.
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