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
Part of the Neuromethods book series (NM, volume 99)


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 



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.).


  1. 1.
    Sunkin SM, Ng L, Lau C et al (2013) Allen Brain Atlas: an integrated spatio-temporal portal for exploring the central nervous system. Nucleic Acids Res 41(Database issue):D996–D1008CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    Itzkovitz S, van Oudenaarden A (2011) Validating transcripts with probes and imaging technology. Nat Methods 8(4 Suppl):S12–S19CrossRefPubMedCentralPubMedGoogle Scholar
  3. 3.
    Hatten ME, Heintz N (2005) Large-scale genomic approaches to brain development and circuitry. Annu Rev Neurosci 28:89–108CrossRefPubMedGoogle Scholar
  4. 4.
    Player AN, Shen LP, Kenny D et al (2001) Single-copy gene detection using branched DNA (bDNA) in situ hybridization. J Histochem Cytochem 49:603–612CrossRefPubMedGoogle Scholar
  5. 5.
    Wang F, Flanagan J, Su N et al (2012) RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn 14:22–29CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Wang Z, Portier BP, Gruver AM et al (2013) Automated quantitative RNA in situ hybridization for resolution of equivocal and heterogeneous ERBB2 (HER2) status in invasive breast carcinoma. J Mol Diagn 15:210–219CrossRefPubMedGoogle Scholar
  7. 7.
    Tubbs RR, Wang H, Wang Z et al (2013) Ultrasensitive RNA in situ hybridization for detection of restricted clonal expression of low abundance immunoglobulin light chain mRNA in B-Cell lymphoproliferative disorders. Am J Clin Pathol 140:736–746CrossRefPubMedGoogle Scholar
  8. 8.
    Mehrad M, Carpenter DH, Chernock RD et al (2013) Papillary squamous cell carcinoma of the head and neck: clinicopathologic and molecular features with special reference to human papillomavirus. Am J Surg Pathol 37:1349–1356CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Schache AG, Liloglou T, Risk JM et al (2013) Validation of a novel diagnostic standard in HPV-positive oropharyngeal squamous cell carcinoma. Br J Cancer 108:1332–1339CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Bishop JA, Ma XJ, Wang H et al (2012) Detection of transcriptionally active high-risk HPV in patients with head and neck squamous cell carcinoma as visualized by a novel E6/E7 mRNA in situ hybridization method. Am J Surg Pathol 36:1874–1882CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Liu X, Bates R, Yin DM et al (2011) Specific regulation of NRG1 isoform expression by neuronal activity. J Neurosci 31:8491–8501CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Hickman S, Kingery N, Ohsumi T et al (2013) The microglial sensome revealed by direct RNA sequencing. Nat Neurosci 16(12):1896–1905CrossRefPubMedGoogle Scholar
  13. 13.
    Bordeaux JM, Cheng H, Welsh AW et al (2012) Quantitative in situ measurement of estrogen receptor mRNA predicts response to tamoxifen. PLoS One 7:e36559CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Lewis JS Jr, Chernock RD, Ma XJ et al (2012) Partial p16 staining in oropharyngeal squamous cell carcinoma: extent and pattern correlate with human papillomavirus RNA status. Mod Pathol 25:1212–1220CrossRefPubMedGoogle Scholar
  15. 15.
    Payne RE, Wang F, Su N et al (2012) Viable circulating tumour cell detection using multiplex RNA in situ hybridisation predicts progression-free survival in metastatic breast cancer patients. Br J Cancer 106:1790–1797CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Ukpo OC, Flanagan JJ, Ma XJ et al (2011) High-risk human papillomavirus E6/E7 mRNA detection by a novel in situ hybridization assay strongly correlates with p16 expression and patient outcomes in oropharyngeal squamous cell carcinoma. Am J Surg Pathol 35:1343–1350CrossRefPubMedGoogle Scholar
  17. 17.
    Staudt ND, Jo M, Hu J et al (2013) Myeloid cell receptor LRP1/CD91 regulates monocyte recruitment and angiogenesis in tumors. Cancer Res 73:3902–3912CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Burd CE, Sorrentino JA, Clark KS et al (2013) Monitoring tumorigenesis and senescence in vivo with a p16INK4a-luciferase model. Cell 152:340–351CrossRefPubMedCentralPubMedGoogle Scholar
  19. 19.
    Shames DS, Carbon J, Walter K et al (2013) High heregulin expression is associated with activated HER3 and may define an actionable biomarker in patients with squamous cell carcinomas of the head and neck. PLoS One 8:e56765CrossRefPubMedCentralPubMedGoogle Scholar
  20. 20.
    Gao G, Chernock RD, Gay HA et al (2013) A novel RT-PCR method for quantification of human papillomavirus transcripts in archived tissues and its application in oropharyngeal cancer prognosis. Int J Cancer 132:882–890CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    Kim MA, Jung JE, Lee HE et al (2013) In situ analysis of HER2 mRNA in gastric carcinoma: comparison with fluorescence in situ hybridization, dual-color silver in situ hybridization, and immunohistochemistry. Hum Pathol 44:487–494CrossRefPubMedGoogle Scholar
  22. 22.
    Ziskin JL, Dunlap D, Yaylaoglu M et al (2013) In situ validation of an intestinal stem cell signature in colorectal cancer. Gut 62:1012–1023CrossRefPubMedGoogle Scholar
  23. 23.
    Warrick JI, Tomlins SA, Carskadon SL et al (2014) Evaluation of tissue PCA3 expression in prostate cancer by RNA in situ hybridization-a correlative study with urine PCA3 and TMPRSS2-ERG. Mod Pathol 27(4):609–20CrossRefPubMedCentralPubMedGoogle Scholar
  24. 24.
    van Beelen GA, Østvik AE, Brenna Ø et al (2013) REG gene expression in inflamed and healthy colon mucosa explored by in situ hybridization. Cell Tissue Res 352:639–646CrossRefGoogle Scholar
  25. 25.
    Ouwendijk WJ, Abendroth A, Traina-Dorge V et al (2013) T-cell infiltration correlates with CXCL10 expression in ganglia of cynomolgus macaques with reactivated simian varicella virus. J Virol 87:2979–2982CrossRefPubMedCentralPubMedGoogle Scholar
  26. 26.
    Sørdal Ø, Qvigstad G, Nordrum IS et al (2013) In situ hybridization in human and rodent tissue by the use of a new and simplified method. Appl Immunohistochem Mol Morphol 21:185–189PubMedGoogle Scholar
  27. 27.
    Takata S, Sawa Y, Uchiyama T et al (2013) Expression of Toll-Like receptor 4 in glomerular endothelial cells under diabetic conditions. Acta Histochem Cytochem 46:35–42CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Hanley MB, Lomas W, Mittar D et al (2013) Detection of low abundance RNA molecules in individual cells by flow cytometry. PLoS One 8:e57002CrossRefPubMedCentralPubMedGoogle Scholar
  29. 29.
    Shinohara DB, Vaghasia AM, Yu SH et al (2013) A mouse model of chronic prostatic inflammation using a human prostate cancer-derived isolate of Propionibacterium acnes. Prostate 73:1007–1015CrossRefPubMedCentralPubMedGoogle Scholar
  30. 30.
    Safronetz D, Prescott J, Haddock E et al (2013) Hamster-adapted Sin Nombre virus causes disseminated infection and efficiently replicates in pulmonary endothelial cells without signs of disease. J Virol 87:4778–4782CrossRefPubMedCentralPubMedGoogle Scholar
  31. 31.
    Brenna Ø, Furnes MW, Drozdov I et al (2013) Relevance of TNBS-colitis in rats: a methodological study with endoscopic, histologic and Transcriptomic characterization and correlation to IBD. PLoS One 8:e54543CrossRefPubMedCentralPubMedGoogle Scholar
  32. 32.
    Barry ER, Morikawa T, Butler BL et al (2013) Restriction of intestinal stem cell expansion and the regenerative response by YAP. Nature 493:106–110CrossRefPubMedCentralPubMedGoogle Scholar
  33. 33.
    Yan KS, Chia LA, Li X et al (2012) The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations. Proc Natl Acad Sci U S A 109:466–471CrossRefPubMedCentralPubMedGoogle Scholar

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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