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

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Transglutaminases
protein-glutamine gamma-glutamyltransferase
in situ hybridization
In Situ Hybridization
RNA probes
RNA Probes
haptens
Haptens
Messenger RNA
RNA
Enzymes
hybridization
enzymes
Glutaminase
antisense RNA
Antisense RNA
antibodies
dipeptides
Antibodies
Dipeptides

All Science Journal Classification (ASJC) codes

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

Cite this

Transglutaminase-mediated in situ hybridization (transish) for mRNA detection in mammalian tissues. / Miyawaki, Katsuyuki; Noji, Sumihare; Kamiya, Noriho.

In Situ Hybridization Methods. Springer New York, 2015. p. 549-558.

Research output: Chapter in Book/Report/Conference proceedingChapter

Miyawaki, Katsuyuki ; Noji, Sumihare ; Kamiya, Noriho. / Transglutaminase-mediated in situ hybridization (transish) for mRNA detection in mammalian tissues. In Situ Hybridization Methods. Springer New York, 2015. pp. 549-558
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