Advertisement

Quantitative 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
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

In situ hybridization Nucleic acid hybridization Messenger RNA RNAscope Noncoding RNA Cancer Gene expression Biomarker 

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.).

References

  1. 1.
    Itzkovitz S, van Oudenaarden A (2011) Validating transcripts with probes and imaging technology. Nat Methods 8:S12–S19PubMedCrossRefPubMedCentralGoogle Scholar
  2. 2.
    ENCODE Project Consortium (2012) An integrated encyclopedia of DNA elements in the human genome. Nature 489:57–74CrossRefGoogle Scholar
  3. 3.
    Djebali S, Davis CA, Merkel A et al (2012) Landscape of transcription in human cells. Nature 489:101–108PubMedCrossRefPubMedCentralGoogle Scholar
  4. 4.
    Mercer TR, Mattick JS (2013) Structure and function of long noncoding RNAs in epigenetic regulation. Nat Struct Mol Biol 20:300–307PubMedCrossRefGoogle Scholar
  5. 5.
    Ulitsky I, Bartel DP (2013) lincRNAs: genomics, evolution, and mechanisms. Cell 154:26–46PubMedCrossRefPubMedCentralGoogle Scholar
  6. 6.
    Cheetham SW, Gruhl F, Mattick JS et al (2013) Long noncoding RNAs and the genetics of cancer. Br J Cancer 108:2419–2425PubMedCrossRefPubMedCentralGoogle Scholar
  7. 7.
    Gupta RA, Shah N, Wang KC et al (2010) Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature 464:1071–1076PubMedCrossRefPubMedCentralGoogle Scholar
  8. 8.
    Player AN, Shen LP, Kenny D et al (2001) Single-copy gene detection using branched DNA (bDNA) in situ hybridization. J Histochem Cytochem 49:603–612PubMedCrossRefGoogle Scholar
  9. 9.
    Nuovo GJ (1995) In situ PCR: protocols and applications. Genome Res 4:S151–S167CrossRefGoogle Scholar
  10. 10.
    Speel EJ, Saremaslani P, Roth J et al (1998) Improved mRNA in situ hybridization on formaldehyde-fixed and paraffin-embedded tissue using signal amplification with different haptenized tyramides. Histochem Cell Biol 110:571–577PubMedCrossRefGoogle Scholar
  11. 11.
    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 14:22–29PubMedCrossRefPubMedCentralGoogle Scholar
  12. 12.
    Tanas MR, Sboner A, Oliveira AM et al (2011) Identification of a disease-defining gene fusion in epithelioid hemangioendothelioma. Sci Transl Med 3:98ra82PubMedCrossRefGoogle 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:e36559PubMedCrossRefPubMedCentralGoogle Scholar
  14. 14.
    Hanley MB, Lomas W, Mittar D et al (2013) Detection of low abundance RNA molecules in individual cells by flow cytometry. PLoS One 8:e57002PubMedCrossRefPubMedCentralGoogle Scholar
  15. 15.
    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 15:210–219PubMedCrossRefGoogle Scholar
  16. 16.
    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 140:736–746PubMedCrossRefGoogle Scholar
  17. 17.
    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 37:1349–1356PubMedCrossRefPubMedCentralGoogle Scholar
  18. 18.
    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 108:1332–1339PubMedCrossRefPubMedCentralGoogle Scholar
  19. 19.
    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 36:1874–1882PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    Liu X, Bates R, Yin DM et al (2011) Specific regulation of NRG1 isoform expression by neuronal activity. J Neurosci 31:8491–8501PubMedCrossRefPubMedCentralGoogle Scholar
  21. 21.
    Hickman S, Kingery N, Ohsumi T et al (2013) The microglial Sensome revealed by direct RNA sequencing. Nat Neurosci 16:1896–1905PubMedCrossRefGoogle Scholar
  22. 22.
    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 25:1212–1220PubMedCrossRefGoogle Scholar
  23. 23.
    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 106:1790–1797PubMedCrossRefPubMedCentralGoogle Scholar
  24. 24.
    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 35:1343–1350PubMedCrossRefGoogle Scholar
  25. 25.
    Staudt ND, Jo M, Hu J et al (2013) Myeloid cell receptor LRP1/CD91 regulates monocyte recruitment and angiogenesis in tumors. Cancer Res 73:3902–3912PubMedCrossRefPubMedCentralGoogle Scholar
  26. 26.
    Burd CE, Sorrentino JA, Clark KS et al (2013) Monitoring tumorigenesis and senescence in vivo with a p16INK4a-luciferase model. Cell 152:340–351PubMedCrossRefPubMedCentralGoogle Scholar
  27. 27.
    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:e56765PubMedCrossRefPubMedCentralGoogle Scholar
  28. 28.
    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 132:882–890PubMedCrossRefPubMedCentralGoogle Scholar
  29. 29.
    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 44:487–494PubMedCrossRefGoogle Scholar
  30. 30.
    Ziskin JL, Dunlap D, Yaylaoglu M et al (2013) In situ validation of an intestinal stem cell signature in colorectal cancer. Gut 62:1012–1023PubMedCrossRefGoogle Scholar
  31. 31.
    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 27:609–620Google Scholar
  32. 32.
    van Beelen Granlund A, Østvik AE, Brenna Ø et al (2013) REG gene expression in inflamed and healthy colon mucosa explored by in situ hybridization. Cell Tissue Res 352:639–646PubMedCrossRefPubMedCentralGoogle Scholar
  33. 33.
    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 87:2979–2982PubMedCrossRefPubMedCentralGoogle Scholar
  34. 34.
    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
  35. 35.
    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 46:35–42PubMedCrossRefPubMedCentralGoogle Scholar
  36. 36.
    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 73:1007–1015PubMedCrossRefPubMedCentralGoogle Scholar
  37. 37.
    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 87:4778–4782PubMedCrossRefPubMedCentralGoogle Scholar
  38. 38.
    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:e54543PubMedCrossRefPubMedCentralGoogle Scholar
  39. 39.
    Barry ER, Morikawa T, Butler BL et al (2013) Restriction of intestinal stem cell expansion and the regenerative response by YAP. Nature 493:106–110PubMedCrossRefPubMedCentralGoogle Scholar
  40. 40.
    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 109:466–471PubMedCrossRefPubMedCentralGoogle Scholar
  41. 41.
    Hammond ME, Hayes DF, Dowsett M et al (2010) American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. Arch Pathol Lab Med 134:907–922PubMedPubMedCentralGoogle Scholar

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.3960 Point Eden WayHaywardUSA

Personalised recommendations