mRNA-targeted Fluorescent in Situ Hybridization (FISH) of Gram-negative Bacteria Without Template Amplification or Tyramide Signal Amplification

Journal Article · Sat Dec 01 00:00:00 EST 2007 · Journal of Microbiological Methods, 71(3):246-255

DOI:https://doi.org/10.1016/j.mimet.2007.09.011· OSTI ID:924053

Coleman, James R; Culley, David E; Chrisler, William B; Brockman, Fred J

Technologies are needed to study gene expression at the level of individual cells within a population or microbial community. Fluorescent in situ hybridization (FISH) supplies high-resolution spatial information and has been widely applied to study microbial communities at the rRNA level. While mRNA-targeted FISH has been popular for studying gene expression in eukaryotic cells, very little success has been achieved with prokaryotes. At present, detection of specific mRNAs in individual prokaryotic cells requires the use of in situ-RT-PCR or tyramide signal amplification (TSA). In this study we used DNA oligonucleotide probes labeled with a single near-infrared dye in FISH assays to detect multicopy plasmid-based and endogenous mRNA molecules in Escherichia coli and Shewanella oneidensis MR-1. We took advantage of the fact there is much less background signal produced by biological materials and support matrices in the near-infrared spectrum and thus long camera exposure times could be used. In addition, we demonstrate that a combination of probes targeting both rRNA and mRNA could be successfully employed within the same FISH assay. These results, as well as ongoing R&D improvements in NIR and infrared dyes, indicate the FISH approach we demonstrated could be applied in certain environmental settings to monitor gene expression in mixed populations.

• Research Organization: Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
• Sponsoring Organization: USDOE
• DOE Contract Number: AC05-76RL01830
• OSTI ID: 924053
• Report Number(s): PNNL-SA-55589; JMIMDQ; KP1102010; TRN: US200805%%102
• Journal Information: Journal of Microbiological Methods, 71(3):246-255, Vol. 71, Issue 3; ISSN 0167-7012
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
AMPLIFICATION
BACTERIA
BIOLOGICAL MATERIALS
CAMERAS
COMMUNITIES
DETECTION
DNA
DYES
ESCHERICHIA COLI
GENES
IN-SITU HYBRIDIZATION
MATRICES
MONITORS
OLIGONUCLEOTIDES
PROBES
Florescent in situ hybridization(FISH)
near-infrared(NIR)
Tyramide signal amplification(TSA)
green fluorescent protein(GFP)
mRNA