Visualization of mcr mRNA in a methanogen by fluorescence in situ hybridization with an oligonucleotide probe and two-pass tyramide signal amplification (two-pass TSA-FISH)

Kengo Kubota, Akiyoshi Ohashi, Hiroyuki Imachi, Hideki Harada

Research output: Contribution to journalArticlepeer-review

31 Citations (Scopus)

Abstract

Two-pass tyramide signal amplification-fluorescence in situ hybridization (two-pass TSA-FISH) with a horseradish peroxidase (HRP)-labeled oligonucleotide probe was applied to detect prokaryotic mRNA. In this study, mRNA of a key enzyme for methanogenesis, methyl coenzyme M reductase (mcr), in Methanococcus vannielii was targeted. Applicability of mRNA-targeted probes to in situ hybridization was verified by Clone-FISH. It was observed that sensitivity of two-pass TSA-FISH was significantly higher than that of TSA-FISH, which was further increased by the addition of dextran sulphate in TSA working solution. Signals from two-pass TSA-FISH were more reliable compared to the weak, spotty signals yielded by TSA-FISH.

Original languageEnglish
Pages (from-to)521-528
Number of pages8
JournalJournal of Microbiological Methods
Volume66
Issue number3
DOIs
Publication statusPublished - 2006 Sep 1

Keywords

  • Fluorescence in situ hybridization (FISH)
  • Methanococcus vannielii
  • Methanogen
  • Methyl coenzyme M reductase (mcr)
  • Tyramide signal amplification (TSA)
  • mRNA

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology
  • Microbiology (medical)

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