Simultaneous fluorescence in situ hybridization of mRNA and rRNA for the detection of gene expression in environmental microbes

Methods Enzymol. 2005;397:352-71. doi: 10.1016/S0076-6879(05)97021-3.

Abstract

A protocol is presented for the detection of gene expression in environmental microorganisms by means of fluorescence in situ hybridization (FISH). Messenger RNA (mRNA) is hybridized with digoxigenin (DIG)- or fluorescein (FLUOS)-labeled ribonucleotide probes. Subsequently the hybrid is detected immunochemically with a horseradish peroxidase (HRP)-labeled antibody and tyramide signal amplification (catalyzed reporter deposition, CARD). After mRNA FISH, microorganisms can be identified by rRNA FISH with oligonucleotide probes labeled either with a fluorochrome or with HRP. Sample preparation and cell permeabilization strategies for various microbial cell types are discussed. The synthesis of DIG- and FLUOS-labeled probes, as well as custom labeling of tyramides with different fluorochromes, is described. As a case study, we describe in detail mRNA FISH of the particulate methane-monooxygenase, subunit A (pmoA) in endosymbiotic bacteria from tissue sections of a marine mollusc. PmoA is used as a marker gene for methanotrophy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Digoxigenin / chemistry
  • Environmental Microbiology*
  • Fluorescein / chemistry
  • Gene Expression
  • In Situ Hybridization, Fluorescence / methods*
  • Mytilidae / microbiology
  • Oxygenases / biosynthesis
  • Oxygenases / genetics
  • RNA Probes / chemistry
  • RNA, Messenger / analysis*
  • RNA, Ribosomal / analysis*

Substances

  • RNA Probes
  • RNA, Messenger
  • RNA, Ribosomal
  • Oxygenases
  • methane monooxygenase
  • Digoxigenin
  • Fluorescein