期刊
DEVELOPMENT
卷 143, 期 15, 页码 2862-2867出版社
COMPANY OF BIOLOGISTS LTD
DOI: 10.1242/dev.138560
关键词
CLARITY; Amplification; Single-molecule RNA
资金
- Heritage Medical Research Institute [HMRI-15-09-01]
- National Institutes of Health [1DP2OD017782, 5R01EB006192, R01 HD075605, 1DP2OD008530]
- Caltech Amgen Chem-BioEngineering award [CBEA] [7200811262]
- Gordon and Betty Moore Foundation [GBMF2809]
- National Science Foundation Molecular Programming Project [NSF-CCF-1317694]
- John Simon Guggenheim Memorial Foundation
- McKnight Foundation
- Beckman Institute at Caltech [PMTC]
- Beckman Institute at Caltech [CLOVER]
- Beckman Institute at Caltech [pilot center]
Accurate and robust detection of mRNA molecules in thick tissue samples can reveal gene expression patterns in single cells within their native environment. Preserving spatial relationships while accessing the transcriptome of selected cells is a crucial feature for advancing many biological areas - from developmental biology to neuroscience. However, because of the high autofluorescence background of many tissue samples, it is difficult to detect single-molecule fluorescence in situ hybridization (smFISH) signals robustly in opaque thick samples. Here, we draw on principles from the emerging discipline of dynamic nucleic acid nanotechnology to develop a robust method for multi-color, multi-RNA imaging in deep tissues using single-molecule hybridization chain reaction (smHCR). Using this approach, single transcripts can be imaged using epifluorescence, confocal or selective plane illumination microscopy (SPIM) depending on the imaging depth required. We show that smHCR has high sensitivity in detecting mRNAs in cell culture and whole-mount zebrafish embryos, and that combined with SPIM and PACT (passive CLARITY technique) tissue hydrogel embedding and clearing, smHCR can detect single mRNAs deep within thick (0.5 mm) brain slices. By simultaneously achieving similar to 20-fold signal amplification and diffraction-limited spatial resolution, smHCR offers a robust and versatile approach for detecting single mRNAs in situ, including in thick tissues where high background undermines the performance of unamplified smFISH.
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