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

22 Citations (Scopus)

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
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages43-59
Number of pages17
Volume980
ISBN (Print)9781627032865
DOIs
StatePublished - 2013
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume980
ISSN (Print)10643745

Fingerprint

MicroRNAs
Pancreatic Neoplasms
In Situ Hybridization
Adenocarcinoma
Paraffin
Formaldehyde
Nucleic Acid Probes
Untranslated RNA
Horseradish Peroxidase
Fluorescein
Fluorescent Dyes
Neoplasms
Fluorescence
Biopsy
Proteins

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)

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 Methods in Molecular Biology (Vol. 980, pp. 43-59). (Methods in Molecular Biology; Vol. 980). Humana Press Inc.. https://doi.org/10.1007/978-1-62703-287-2_4

A method for conducting highly sensitive MicroRNA in situ hybridization and immunohistochemical analysis in pancreatic cancer. / Sempere, Lorenzo F.; Korc, Murray.

Methods in Molecular Biology. Vol. 980 Humana Press Inc., 2013. p. 43-59 (Methods in Molecular Biology; Vol. 980).

Research output: Chapter in Book/Report/Conference proceedingChapter

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