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

Katsuyuki Miyawaki, Sumihare Noji, Noriho Kamiya

研究成果: 書籍/レポート タイプへの寄稿

抄録

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.

本文言語英語
ホスト出版物のタイトルIn Situ Hybridization Methods
出版社Springer New York
ページ549-558
ページ数10
ISBN(電子版)9781493923038
ISBN(印刷版)9781493923021
DOI
出版ステータス出版済み - 2月 18 2015

!!!All Science Journal Classification (ASJC) codes

  • 医学(全般)
  • 神経科学(全般)
  • 農業および生物科学(全般)

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