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A RNAscope whole mount approach that can be combined with immunofluorescence to quantify differential distribution of mRNA

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Abstract

RNAscope® technology provided by Advanced Cell Diagnostics (ACD) allows the detection and evaluation of coinciding mRNA expression profiles in the same or adjacent cells in unprecedented quantitative detail using multicolor fluorescent in situ hybridization (FISH). While already extensively used in thinly sectioned material of various pathological tissues and, to a lesser extent, in some whole mounts, we provide here a detailed approach to use the fluorescent RNAscope method in the mouse inner ear and thick brain sections by modifying and adapting existing techniques of whole mount fluorescent in situ hybridization (WH-FISH). We show that RNAscope WH-FISH can be used to quantify local variation in overlaying mRNA expression intensity, such as neurotrophin receptors along the length of the mouse cochlea. We also show how RNAscope WH-FISH can be combined with immunofluorescence (IF) of some epitopes that remain after proteinase digestion and, to some extent, with fluorescent protein markers such as tdTomato. Our WH-FISH technique provides an approach to detect cell-specific quantitative differences in developing and mature adjacent cells, an emerging issue revealed by improved cellular expression profiling. Further, the presented technique may be useful in validating single-cell RNAseq data on expression profiles in a range of tissue known or suspected to have locally variable mRNA expression levels.

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Acknowledgments

This work was supported by INI (Roy J. Carver Chair in Neuroscience), R01 (DC005590), the University of Iowa (sabbatical to BF) and Decibel Therapeutics. The authors express their gratitude to Dr. David Ginty (Harvard University) for providing the TrkC-tdTomato-expressing mice used for Fig. 3.

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Authors and Affiliations

  1. Department of Biology, University of Iowa, Iowa City, IA, 52242, USA

    Jennifer Kersigo & Bernd Fritzsch

  2. Decibel Therapeutics, Boston, MA, USA

    Ning Pan & Immaculada Silos-Santiago

  3. Iowa Neuroscience Institute, University of Iowa, Iowa City, IA, USA

    Joseph D. Lederman, Snehajyoti Chatterjee & Ted Abel

  4. Institute of Biotechnology CAS, Vestec, Czechia

    Gabriela Pavlinkova

Authors
  1. Jennifer Kersigo
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  2. Ning Pan
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  3. Joseph D. Lederman
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  4. Snehajyoti Chatterjee
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  5. Ted Abel
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  6. Gabriela Pavlinkova
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  7. Immaculada Silos-Santiago
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  8. Bernd Fritzsch
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Corresponding author

Correspondence to Bernd Fritzsch.

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The University of Iowa, Decibel Therapeutics, or Harvard University Institutional Animal Care and Use Committee reviewed and approved all procedures used here.

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Kersigo, J., Pan, N., Lederman, J.D. et al. A RNAscope whole mount approach that can be combined with immunofluorescence to quantify differential distribution of mRNA. Cell Tissue Res 374, 251–262 (2018). https://doi.org/10.1007/s00441-018-2864-4

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  • DOI: https://doi.org/10.1007/s00441-018-2864-4

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