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Published August 1, 2016 | Published
Journal Article Open

Single-molecule RNA detection at depth via hybridization chain reaction and tissue hydrogel embedding and clearing


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 ∼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.

Additional Information

© 2016 Published by The Company of Biologists Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. Received April 11, 2016. Accepted June 20, 2016. Author Contributions: The project was designed by VG, NP, and LC. Experiments were performed by SS and EL (cultured cells), MS (whole-mount zebrafish embryos), and T-FH and AG (PACT-cleared adult mouse brain sections). Automated dot classification analyses were performed by SS and EL. AG built the SPIM for PACT (with help from AL) and performed SPIM/PACT imaging and all video renderings. All authors contributed to protocol optimization, designed experiments, analyzed data, and edited the manuscript. Funding: This work was supported by a Heritage Principal Investigatorship (VG), an NIH Director's New Innovator IDP20D017782-01 and PECASE (VG), the Beckman Institute at Caltech (CLOVER: CLARITY, Optogenetics, and Vector Engineering Research Center), a Caltech-CBEA, the NIH (5R01EB006192), the Gordon and Betty Moore Foundation (GBMF2809), the NSF Molecular Programming Project (NSF-CCF-1317694), the Beckman Institute at Caltech (Programmable Molecular Technology Center), and a Guggenheim Fellowship (NAP), the NIH (R01 HD075605 and 1DP2OD008530 to LC), a Beckman Institute pilot center (LC), and the McKnight Foundation (LC).

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