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Pancreatic Cancer pp 43–59Cite as

A Method for Conducting Highly Sensitive MicroRNA In Situ Hybridization and Immunohistochemical Analysis in Pancreatic Cancer

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 980))

Abstract

Profiling experiments in whole tissue biopsies have linked altered expression of microRNAs (miRNAs) to different types of cancer, including pancreatic ductal adenocarcinoma (PDAC). Emerging evidence indicates that altered miRNA expression can occur in different cellular compartments (cancer and non-cancer cells) in tumor lesions, and thus it is important to ascertain which specific cell type expresses a particulars miRNA in PDAC tissues. Here, we describe a highly sensitive fluorescence-based ISH method to visualize miRNA accumulation within individual cells in formalin-fixed paraffin-embedded (FFPE) tissue sections using 5′ and 3′ terminally fluorescein-labeled locked nucleic acid (LNA)-modified probes. We describe a multicolor ISH/IHC method based on sequential rounds of horseradish peroxidase (HRP)-mediated tyramide signal amplification (TSA) reactions with different in-house synthesized fluorochrome-conjugated substrates that enable co-detection of miRNAs, abundant noncoding RNAs and protein markers for signal quantification, and cell type co-localization studies in FFPE pancreatic tissue sections from clinical specimens and mouse models of PDAC.

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Acknowledgment

This work was supported by National Institutes of Health (NIH) and National Cancer Institute (NCI) grants CA-R37-075059 (M.K.), CA-R21-135664 (M.K), CA-R21-133715 (M.K), and AACR-PanCAN Laurie and Paul MacCaskill Career Development Award for Pancreatic Cancer Research grant 08-20-25-SEMP (L.S.).

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Authors and Affiliations

  1. Department of Medicine, Dartmouth Hitchcock Medical Center, Hanover, NH, USA

    Lorenzo F. Sempere

  2. Department of Medicine, Norris Cotton Cancer Center, Dartmouth Hitchcock Medical Center, Lebanon, NH, USA

    Murray Korc

  3. Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH, USA

    Murray Korc

Authors
  1. Lorenzo F. Sempere
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  2. Murray Korc
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Correspondence to Murray Korc .

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Editors and Affiliations

  1. , Irving Cancer Research Center, Columbia University, St. Nicholas Avenue 1130, New York, 10032, New York, USA

    Gloria H. Su

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Sempere, L.F., Korc, M. (2013). A Method for Conducting Highly Sensitive MicroRNA In Situ Hybridization and Immunohistochemical Analysis in Pancreatic Cancer. In: Su, G. (eds) Pancreatic Cancer. Methods in Molecular Biology, vol 980. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-287-2_4

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  • DOI: https://doi.org/10.1007/978-1-62703-287-2_4

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-286-5

  • Online ISBN: 978-1-62703-287-2

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