Multiplexed miRNA Fluorescence In Situ Hybridization for Formalin-Fixed Paraffin-Embedded Tissues

  • Neil Renwick
  • Pavol Cekan
  • Claudia Bognanni
  • Thomas TuschlEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1211)


Multiplexed miRNA fluorescence in situ hybridization (miRNA FISH) is an advanced method for visualizing differentially expressed miRNAs, together with other reference RNAs, in archival tissues. Some miRNAs are excellent disease biomarkers due to their abundance and cell-type specificity. However, these short RNA molecules are difficult to visualize due to loss by diffusion, probe mishybridization, and signal detection and signal amplification issues. Here, we describe a reliable and adjustable method for visualizing and normalizing miRNA signals in formalin-fixed paraffin-embedded (FFPE) tissue sections.

Key words

miRNA Fluorescence in situ hybridization Formalin-fixed and paraffin-embedded tissues Molecular diagnostics Multiplexing Signal amplification methods 



N.R. is supported through a K08 award (NS072235) from the National Institute of Neurological Disorders and Stroke. T.T. is an HHMI investigator and supported through R01 funding from NIH CA159227 and MH080442 and a grant by the Starr Cancer Consortium. The project described was supported through the Rockefeller University Bridges to Better Medicine Technology Innovation Fund. The project was also partially supported by Grant Award Number (UL1RR024143) from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH) and NIH Roadmap for Medical Research, and its contents are solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH.


  1. 1.
    Renwick N, Cekan P, Masry PA et al (2013) Multicolor microRNA FISH effectively differentiates tumor types. J Clin Invest 123:2694–2702PubMedCrossRefPubMedCentralGoogle Scholar
  2. 2.
    Hafner M, Renwick N, Farazi TF et al (2012) Barcoded cDNA library preparation for small RNA profiling by next-generation sequencing. Methods 58:164–170PubMedCrossRefPubMedCentralGoogle Scholar
  3. 3.
    Pena JT, Sohn-Lee C, Rouhanifard SH et al (2009) miRNA in situ hybridization in formaldehyde and EDC-fixed tissues. Nat Methods 6:139–141PubMedCrossRefPubMedCentralGoogle Scholar
  4. 4.
  5. 5.
  6. 6.
    Hopman AH, Ramaekers FC, Speel EJ (1998) Rapid synthesis of biotin-, digoxigenin-, trinitrophenyl-, and fluorochrome-labeled tyramides and their application for in situ hybridization using CARD amplification. J Histochem Cytochem 46:771–777PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Neil Renwick
    • 1
  • Pavol Cekan
    • 1
  • Claudia Bognanni
    • 1
  • Thomas Tuschl
    • 1
    Email author
  1. 1.Howard Hughes Medical Institute, Laboratory of RNA Molecular BiologyThe Rockefeller UniversityNew YorkUSA

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