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Single-molecule RNA detection at depth by 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 –...

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Autores principales: Shah, Sheel, Lubeck, Eric, Schwarzkopf, Maayan, He, Ting-Fang, Greenbaum, Alon, Sohn, Chang Ho, Lignell, Antti, Choi, Harry M. T., Gradinaru, Viviana, Pierce, Niles A., Cai, Long
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Company of Biologists Ltd 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5004914/
https://www.ncbi.nlm.nih.gov/pubmed/27342713
http://dx.doi.org/10.1242/dev.138560
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author Shah, Sheel
Lubeck, Eric
Schwarzkopf, Maayan
He, Ting-Fang
Greenbaum, Alon
Sohn, Chang Ho
Lignell, Antti
Choi, Harry M. T.
Gradinaru, Viviana
Pierce, Niles A.
Cai, Long
author_facet Shah, Sheel
Lubeck, Eric
Schwarzkopf, Maayan
He, Ting-Fang
Greenbaum, Alon
Sohn, Chang Ho
Lignell, Antti
Choi, Harry M. T.
Gradinaru, Viviana
Pierce, Niles A.
Cai, Long
author_sort Shah, Sheel
collection PubMed
description 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.
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spelling pubmed-50049142016-09-13 Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing Shah, Sheel Lubeck, Eric Schwarzkopf, Maayan He, Ting-Fang Greenbaum, Alon Sohn, Chang Ho Lignell, Antti Choi, Harry M. T. Gradinaru, Viviana Pierce, Niles A. Cai, Long Development Techniques and Resources 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. The Company of Biologists Ltd 2016-08-01 /pmc/articles/PMC5004914/ /pubmed/27342713 http://dx.doi.org/10.1242/dev.138560 Text en © 2016. Published by The Company of Biologists Ltd http://creativecommons.org/licenses/by/3.0 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.
spellingShingle Techniques and Resources
Shah, Sheel
Lubeck, Eric
Schwarzkopf, Maayan
He, Ting-Fang
Greenbaum, Alon
Sohn, Chang Ho
Lignell, Antti
Choi, Harry M. T.
Gradinaru, Viviana
Pierce, Niles A.
Cai, Long
Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing
title Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing
title_full Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing
title_fullStr Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing
title_full_unstemmed Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing
title_short Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing
title_sort single-molecule rna detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing
topic Techniques and Resources
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5004914/
https://www.ncbi.nlm.nih.gov/pubmed/27342713
http://dx.doi.org/10.1242/dev.138560
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