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Multiplex Fluorescent RNA In Situ Hybridization Via RNAscope

  • Hongwei Wang
  • Nan Su
  • Li-Chong Wang
  • Xingyong Wu
  • Son Bui
  • Kuang-Jung Chang
  • Allissa Nielsen
  • Hong-Thuy Vo
  • Yuling Luo
  • Xiao-Jun Ma
Protocol
Part of the Neuromethods book series (NM, volume 99)

Abstract

Multiplex fluorescent in situ hybridization is a useful tool for neurobiology applications. We have developed a novel RNA ISH technology, RNAscope, with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. Here, we present a detailed protocol of Multiplex Fluorescent RNAscope Assay that permits visualization of three target genes simultaneously on fresh frozen brain tissue sections. The step-by-step protocol describes tissue preparation, fixation, pretreatment, probe hybridization, signal amplification, and visualization. The Multiplex Fluorescent RNAscope Assay is particularly useful for detecting low-copy genes, as it offers high sensitivity and specificity. We also discuss critical steps for ensuring successful experiments.

Key words

In situ hybridization/methods Nucleic acid hybridization/methods RNA, messenger/analysis RNAscope/methods Multiplex fluorescent/methods Gene expression Brain Central nervous system Neurobiology Mouse 

Notes

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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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Hongwei Wang
    • 1
  • Nan Su
    • 1
  • Li-Chong Wang
    • 1
  • Xingyong Wu
    • 1
  • Son Bui
    • 1
  • Kuang-Jung Chang
    • 1
  • Allissa Nielsen
    • 1
  • Hong-Thuy Vo
    • 1
  • Yuling Luo
    • 1
  • Xiao-Jun Ma
    • 1
  1. 1.Advanced Cell Diagnostics, Inc.HaywardUSA

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