Dual-Color Ultrasensitive Bright-Field RNA In Situ Hybridization with RNAscope

  • Hongwei Wang
  • Nan Su
  • Li-Chong Wang
  • Xingyong Wu
  • Son Bui
  • Allissa Nielsen
  • Hong-Thuy Vo
  • Yuling Luo
  • Xiao-Jun MaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1211)


In situ hybridization (ISH) techniques have been important to the study of gene expression signatures in cells and tissues. The ability to detect multiple targets simultaneously is especially valuable, since it allows dissecting gene expression of distinct cell types with precise cellular and subcellular resolution within morphological context. Recently, we have reported using a novel dual-color ultrasensitive bright-field RNA in situ hybridization for detection of clonally restricted immunoglobulin light chain mRNA expression in B cell lymphomas. Here, we present detailed protocols of RNAscope 2-Plex assays for FFPE tissue sections. The protocols describe the tissue preparation, pretreatment, probe hybridization, signal amplification, visualization, and analysis, as well as emphasize the critical steps for ensuring successful staining.

Key words

In situ hybridization Nucleic acid hybridization RNA Messenger RNAscope Cancer Dual-color CISH Gene expression Biomarker 



Supported in part by grants from the NIH (R43/44CA122444 to Y.L. and R43CA168019 to X.-J. M.) 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 2014

Authors and Affiliations

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

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