Protocol abstract

Nature Protocols 2, 2520 - 2528 (2007)
Published online: 11 October 2007 | doi:10.1038/nprot.2007.313

Subject Categories: Imaging | Cell and developmental biology

Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification

Asli N Silahtaroglu1, Dorrit Nolting2, Lars Dyrskjøt3, Eugene Berezikov4, Morten Møller5, Niels Tommerup1 & Sakari Kauppinen1,6

The ability to determine spatial and temporal microRNA (miRNA) accumulation at the tissue, cell and subcellular levels is essential for understanding the biological roles of miRNAs and miRNA-associated gene regulatory networks. This protocol describes a method for fast and effective detection of miRNAs in frozen tissue sections using fluorescence in situ hybridization (FISH). The method combines the unique miRNA recognition properties of locked nucleic acid (LNA)-modified oligonucleotide probes with FISH using the tyramide signal amplification (TSA) technology. Although both approaches have previously been shown to increase detection sensitivity in FISH, combining these techniques into one protocol significantly decreases the time needed for miRNA detection in cryosections, while simultaneously retaining high detection sensitivity. Starting with fixation of the tissue sections, this miRNA FISH protocol can be completed within approximately 6 h and allows miRNA detection in a wide variety of animal tissue cryosections as well as in human tumor biopsies at high cellular resolution.

  1. Wilhelm Johannsen Centre for Functional Genome Research, Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
  2. Department of Orthodontics, School of Dentistry, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
  3. Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital, Skejby, DK-8200 Aarhus N, Denmark.
  4. Hubrecht Institute, Uppsalalaan 8, 3584CT Utrecht, The Netherlands.
  5. Department of Neuroscience and Pharmacology, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
  6. Santaris Pharma, Boege Alle 3, DK-2970 Denmark.

Correspondence to: Asli N Silahtaroglu1 e-mail: