A method for conducting highly sensitive MicroRNA in situ hybridization and immunohistochemical analysis in pancreatic cancer

Lorenzo F. Sempere, Murray Korc

Research output: Chapter in Book/Report/Conference proceedingChapter

24 Scopus citations

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 particular 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.

Original languageEnglish (US)
Title of host publicationPancreatic Cancer
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc.
Pages43-59
Number of pages17
ISBN (Print)9781627032865
DOIs
StatePublished - 2013

Publication series

NameMethods in Molecular Biology
Volume980
ISSN (Print)1064-3745

Keywords

  • Fluorescence microscopy
  • Genetically engineered mouse models
  • Immunohistochemistry
  • In situ hybridization
  • Locked nucleic acid
  • MicroRNA
  • Multiplexing
  • Pancreatic adenocarcinoma
  • Pancreatic cancer

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Medicine(all)

Fingerprint Dive into the research topics of 'A method for conducting highly sensitive MicroRNA in situ hybridization and immunohistochemical analysis in pancreatic cancer'. Together they form a unique fingerprint.

  • Cite this

    Sempere, L. F., & Korc, M. (2013). A method for conducting highly sensitive MicroRNA in situ hybridization and immunohistochemical analysis in pancreatic cancer. In Pancreatic Cancer: Methods and Protocols (pp. 43-59). (Methods in Molecular Biology; Vol. 980). Humana Press Inc.. https://doi.org/10.1007/978-1-62703-287-2_4