PMID- 15345429
OWN - NLM
STAT- MEDLINE
DCOM- 20041130
LR  - 20231105
IS  - 0099-2240 (Print)
IS  - 1098-5336 (Electronic)
IS  - 0099-2240 (Linking)
VI  - 70
IP  - 9
DP  - 2004 Sep
TI  - Simultaneous fluorescence in situ hybridization of mRNA and rRNA in environmental 
      bacteria.
PG  - 5426-33
AB  - We developed for Bacteria in environmental samples a sensitive and reliable mRNA 
      fluorescence in situ hybridization (FISH) protocol that allows for simultaneous 
      cell identification by rRNA FISH. Samples were carbethoxylated with 
      diethylpyrocarbonate to inactivate intracellular RNases and pretreated with 
      lysozyme and/or proteinase K at different concentrations. Optimizing the 
      permeabilization of each type of sample proved to be a critical step in avoiding 
      false-negative or false-positive results. The quality of probes as well as a 
      stringent hybridization temperature were determined with expression clones. To 
      increase the sensitivity of mRNA FISH, long ribonucleotide probes were labeled at 
      a high density with cis-platinum-linked digoxigenin (DIG). The hybrid was 
      immunocytochemically detected with an anti-DIG antibody labeled with horseradish 
      peroxidase (HRP). Subsequently, the hybridization signal was amplified by 
      catalyzed reporter deposition with fluorochrome-labeled tyramides. 
      p-Iodophenylboronic acid and high concentrations of NaCl substantially enhanced 
      the deposition of tyramides and thus increased the sensitivity of our approach. 
      After inactivation of the antibody-delivered HRP, rRNA FISH was performed by 
      following routine protocols. To show the broad applicability of our approach, 
      mRNA of a key enzyme of aerobic methane oxidation, particulate methane 
      monooxygenase (subunit A), was hybridized with different types of samples: pure 
      cultures, symbionts of a hydrothermal vent bivalve, and even sediment, one of the 
      most difficult sample types with which to perform successful FISH. By 
      simultaneous mRNA FISH and rRNA FISH, single cells are identified and shown to 
      express a particular gene. Our protocol is transferable to many different types 
      of samples with the need for only minor modifications of fixation and 
      permeabilization procedures.
FAU - Pernthaler, Annelie
AU  - Pernthaler A
AD  - Max-Planck-Institut fur Marine Mikrobiologie, Celsiusstrabetae 1, D-28359 Bremen, 
      Germany. aperntha@mpi-bremen.de
FAU - Amann, Rudolf
AU  - Amann R
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Appl Environ Microbiol
JT  - Applied and environmental microbiology
JID - 7605801
RN  - 0 (Enzyme Inhibitors)
RN  - 0 (RNA, Bacterial)
RN  - 0 (RNA, Messenger)
RN  - 0 (RNA, Ribosomal)
RN  - EC 3.1.- (Ribonucleases)
RN  - LMR3LZG146 (Diethyl Pyrocarbonate)
SB  - IM
MH  - Bacteria/*genetics
MH  - Cell Membrane Permeability
MH  - Diethyl Pyrocarbonate/pharmacology
MH  - Environment
MH  - Enzyme Inhibitors/pharmacology
MH  - In Situ Hybridization, Fluorescence
MH  - Polymerase Chain Reaction
MH  - RNA, Bacterial/*genetics
MH  - RNA, Messenger/*genetics
MH  - RNA, Ribosomal/*genetics
MH  - Ribonucleases/antagonists & inhibitors
PMC - PMC520857
EDAT- 2004/09/04 05:00
MHDA- 2004/12/16 09:00
PMCR- 2004/09/01
CRDT- 2004/09/04 05:00
PHST- 2004/09/04 05:00 [pubmed]
PHST- 2004/12/16 09:00 [medline]
PHST- 2004/09/04 05:00 [entrez]
PHST- 2004/09/01 00:00 [pmc-release]
AID - 70/9/5426 [pii]
AID - 0152-04 [pii]
AID - 10.1128/AEM.70.9.5426-5433.2004 [doi]
PST - ppublish
SO  - Appl Environ Microbiol. 2004 Sep;70(9):5426-33. doi: 
      10.1128/AEM.70.9.5426-5433.2004.