Transglutaminase-mediated in situ hybridization (transish) for mRNA detection in mammalian tissues

Katsuyuki Miyawaki, Sumihare Noji, Noriho Kamiya

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

In the most commonly used in situ hybridization (ISH) procedure, a hapten-labeled antisense nucleic acid (e.g., RNA) probe is employed to hybridize a target mRNA in tissue sections. The hapten-labeled RNA is then detected by a highly specific hapten-antibody interaction; however, it requires laborious immunostaining steps for spatial coloring on tissue sections. To simplify ISH-based mRNA detection systems, we created a new RNA-(enzyme) n conjugate for sensitive detection of mRNA in tissue sections. In the present simple, antibody-free ISH protocol, an antisense RNA probe of interest is first modified with a specific dipeptide substrate of microbial transglutaminase (MTG) by in vitro transcription. Alkaline phosphatase from a hyperthermophile is then covalently linked to the substrate-labeled RNA by MTG-catalyzed sitespecific conjugation. A robust, multi-enzyme-labeled RNA probe enables the direct labeling of a target mRNA in tissue sections with signaling enzymes under harsh hybridization conditions, leading to one-step signal amplification after hybridization. The application of the new trans glutaminase-mediated ISH (TransISH) strategy to mRNA detection in mammalian tissues was demonstrated.

Original languageEnglish
Title of host publicationIn Situ Hybridization Methods
PublisherSpringer New York
Pages549-558
Number of pages10
ISBN (Electronic)9781493923038
ISBN (Print)9781493923021
DOIs
Publication statusPublished - Feb 18 2015

All Science Journal Classification (ASJC) codes

  • Medicine(all)
  • Neuroscience(all)
  • Agricultural and Biological Sciences(all)

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