PMID- 16260302
OWN - NLM
STAT- MEDLINE
DCOM- 20051202
LR  - 20151119
IS  - 0076-6879 (Print)
IS  - 0076-6879 (Linking)
VI  - 397
DP  - 2005
TI  - Simultaneous fluorescence in situ hybridization of mRNA and rRNA for the 
      detection of gene expression in environmental microbes.
PG  - 352-71
AB  - 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.
FAU - Pernthaler, Annelie
AU  - Pernthaler A
AD  - Department of Molecular Ecology, Max Planck Institute for Marine Microbiology, 
      Bremen, Germany.
FAU - Pernthaler, Jakob
AU  - Pernthaler J
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Methods Enzymol
JT  - Methods in enzymology
JID - 0212271
RN  - 0 (RNA Probes)
RN  - 0 (RNA, Messenger)
RN  - 0 (RNA, Ribosomal)
RN  - EC 1.13.- (Oxygenases)
RN  - EC 1.14.13.25 (methane monooxygenase)
RN  - NQ1SX9LNAU (Digoxigenin)
RN  - TPY09G7XIR (Fluorescein)
SB  - IM
MH  - Animals
MH  - Base Sequence
MH  - Digoxigenin/chemistry
MH  - *Environmental Microbiology
MH  - Fluorescein/chemistry
MH  - Gene Expression
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Mytilidae/microbiology
MH  - Oxygenases/biosynthesis/genetics
MH  - RNA Probes/chemistry
MH  - RNA, Messenger/*analysis
MH  - RNA, Ribosomal/*analysis
EDAT- 2005/11/02 09:00
MHDA- 2005/12/13 09:00
CRDT- 2005/11/02 09:00
PHST- 2005/11/02 09:00 [pubmed]
PHST- 2005/12/13 09:00 [medline]
PHST- 2005/11/02 09:00 [entrez]
AID - S0076-6879(05)97021-3 [pii]
AID - 10.1016/S0076-6879(05)97021-3 [doi]
PST - ppublish
SO  - Methods Enzymol. 2005;397:352-71. doi: 10.1016/S0076-6879(05)97021-3.