Pancreatic Cancer

Volume 980 of the series Methods in Molecular Biology pp 43-59


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

  • Lorenzo F. SempereAffiliated withDepartment of Medicine, Dartmouth Hitchcock Medical Center
  • , Murray KorcAffiliated withDepartment of Medicine, Norris Cotton Cancer Center, Dartmouth Hitchcock Medical CenterDepartment of Pharmacology and Toxicology, Dartmouth Medical School Email author 

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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.

Key words

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