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)

J Microbiol Methods. 2006 Sep;66(3):521-8. doi: 10.1016/j.mimet.2006.02.002. Epub 2006 Mar 20.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • Fluorescent Dyes / chemistry
  • Horseradish Peroxidase / chemistry
  • In Situ Hybridization, Fluorescence / methods*
  • Methanococcus / enzymology*
  • Methanococcus / genetics
  • Microscopy, Fluorescence
  • Oligonucleotide Probes
  • Oxidoreductases / genetics*
  • Oxidoreductases / isolation & purification
  • Polymerase Chain Reaction
  • RNA, Messenger / analysis*
  • RNA, Messenger / genetics
  • RNA, Ribosomal, 16S / chemistry
  • RNA, Ribosomal, 16S / genetics
  • Sensitivity and Specificity

Substances

  • DNA, Bacterial
  • Fluorescent Dyes
  • Oligonucleotide Probes
  • RNA, Messenger
  • RNA, Ribosomal, 16S
  • Oxidoreductases
  • Horseradish Peroxidase
  • methyl coenzyme M reductase