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Detecting MicroRNA in Human Cancer Tissues with Fluorescence In Situ Hybridization

  • Zonggao Shi
  • Jeff J. Johnson
  • M. Sharon Stack
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1039)

Abstract

The technique of nucleic acid in situ hybridization is an effective method for identifying the existence and abundance of nucleic acids in tissue sections or cytological preparations. Such a method has the advantage of keeping morphological relationships intact while identifying changes at the molecular level. As a noncoding regulatory RNA, microRNA has been found to intricately control many physiological and pathological conditions. We provide here a representative fluorescence in situ hybridization protocol for microRNA detection, and note commonly used alternatives, and some troubleshooting points. The method described is based on formalin-fixed paraffin-embedded oral cancer tissues but should be broadly applicable to similarly processed tissues of other types of cancer.

Key words

Squamous cell carcinoma microRNA Fluorescence in situ hybridization (FISH) Formalin-fixed paraffin-embedded (FFPE) tissue 

References

  1. 1.
    Lee RC, Feinbaum RL, Ambros V (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75:843–854PubMedCrossRefGoogle Scholar
  2. 2.
    Andorfer CA, Necela BM, Thompson EA, Perez EA (2011) MicroRNA signatures: clinical biomarkers for the diagnosis and treatment of breast cancer. Trends Mol Med 17:313–319PubMedCrossRefGoogle Scholar
  3. 3.
    Qavi AJ, Kindt JT, Bailey RC (2010) Sizing up the future of microRNA analysis. Anal Bioanal Chem 398:2535–2549PubMedCrossRefGoogle Scholar
  4. 4.
    Nielsen BS (2012) MicroRNA in situ hybridization. Methods Mol Biol 822:67–84PubMedCrossRefGoogle Scholar
  5. 5.
    Shi Z, Johnson JJ, Stack MS (2012) Fluorescence in situ hybridization for microRNA detection in archived oral cancer tissues. J Oncol 2012:903581PubMedCrossRefGoogle Scholar
  6. 6.
    McKay JA, Murray GI, Keith WN, McLeod HL (1997) Amplification of fluorescent in situ hybridisation signals in formalin fixed paraffin wax embedded sections of colon tumour using biotinylated tyramide. Mol Pathol 50:322–325PubMedCrossRefGoogle Scholar
  7. 7.
    Bobrow MN, Moen PT Jr (2001) Tyramide signal amplification (TSA) systems for the enhancement of ISH signals in cytogenetics. Curr Protoc Cytom Chapter 8, Unit 8 9Google Scholar
  8. 8.
    Helander KG (1994) Kinetic studies of formaldehyde binding in tissue. Biotech Histochem 69:177–179PubMedCrossRefGoogle Scholar
  9. 9.
    Fox CH, Johnson FB, Whiting J, Roller PP (1985) Formaldehyde fixation. J Histochem Cytochem 33:845–853PubMedCrossRefGoogle Scholar
  10. 10.
    Glud M, Klausen M, Gniadecki R, Rossing M, Hastrup N, Nielsen FC et al (2009) MicroRNA expression in melanocytic nevi: the usefulness of formalin-fixed, paraffin-embedded material for miRNA microarray profiling. J Invest Dermatol 129:1219–1224PubMedCrossRefGoogle Scholar
  11. 11.
    Liu A, Tetzlaff MT, Vanbelle P, Elder D, Feldman M, Tobias JW et al (2009) MicroRNA expression profiling outperforms mRNA expression profiling in formalin-fixed paraffin-embedded tissues. Int J Clin Exp Pathol 2:519–527PubMedGoogle Scholar
  12. 12.
    Nuovo GJ (2010) In situ detection of microRNAs in paraffin embedded, formalin fixed tissues and the co-localization of their putative targets. Methods 52:307–315PubMedCrossRefGoogle Scholar
  13. 13.
    Kloosterman WP, Wienholds E, de Bruijn E, Kauppinen S, Plasterk RH (2006) In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes. Nat Methods 3:27–29PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Zonggao Shi
    • 1
  • Jeff J. Johnson
    • 1
    • 2
  • M. Sharon Stack
    • 1
    • 2
  1. 1.Harper Cancer Research InstituteUniversity of Notre DameNotre DameUSA
  2. 2.Department of Chemistry and BiochemistryUniversity of Notre DameNotre DameUSA

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