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Quantitative Ultrasensitive Bright-Field RNA In Situ Hybridization with RNAscope

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Book cover In Situ Hybridization Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1211))

Abstract

RNA in situ hybridization (ISH) can provide valuable morphological context for molecular markers on one hand and enable morphological analysis in molecular context on the other hand. It has become increasingly important, thanks to increasing interest in new biomarkers and noncoding RNAs in both research and clinical applications. We have developed an ultrasensitive RNA ISH technology, RNAscope, employing a unique probe design strategy that allows target-specific signal amplification while suppressing background noise. This approach enables single RNA molecule detection in formalin-fixed paraffin-embedded (FFPE) specimens under standard bright-field microscopy and is capable of multiplex detection at the single cell level. After staining, target-specific signals appear as punctate dots present in individual cells in well-preserved tissue morphological context, which facilitates both semiquantitative manual scoring and software-assisted quantitative analysis. Here, we present detailed protocols of RNAscope for FFPE tissue sections. The step-by-step protocols describe tissue preparation, pretreatment, probe hybridization, signal amplification, visualization, and analysis. We also highlight the critical steps for ensuring successful staining.

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Acknowledgments

Supported in part by grants from the NIH (R43/44CA122444 to Y.L.) and the Department of Defense (Breast Cancer Research Program grant W81XWH-06-1-0682 to Y.L.).

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Authors and Affiliations

  1. Advanced Cell Diagnostics, Inc., 3960 Point Eden Way, Hayward, CA, 94545, USA

    Hongwei Wang, Nan Su, Li-Chong Wang, Xingyong Wu, Son Bui, Allissa Nielsen, Hong-Thuy Vo, Yuling Luo & Xiao-Jun Ma

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  1. Hongwei Wang
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  2. Nan Su
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  3. Li-Chong Wang
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  4. Xingyong Wu
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  6. Allissa Nielsen
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  7. Hong-Thuy Vo
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  8. Yuling Luo
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  9. Xiao-Jun Ma
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Corresponding author

Correspondence to Xiao-Jun Ma .

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Editors and Affiliations

  1. Molecular Histology, Bioneer A/S, Horsholm, Denmark

    Boye Schnack Nielsen

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© 2014 Springer Science+Business Media New York

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Wang, H. et al. (2014). Quantitative Ultrasensitive Bright-Field RNA In Situ Hybridization with RNAscope. In: Nielsen, B. (eds) In Situ Hybridization Protocols. Methods in Molecular Biology, vol 1211. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1459-3_16

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  • DOI: https://doi.org/10.1007/978-1-4939-1459-3_16

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1458-6

  • Online ISBN: 978-1-4939-1459-3

  • eBook Packages: Springer Protocols

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