Flow cytometric detection of specific gene expression in prokaryotic cells using in situ RT-PCR.
Prokaryotic in situ RT-PCR was coupled with flow cytometry to detect mRNA transcripts of the toluene dioxygenase (todC1) gene in intact cells of the bacterium Pseudomonas putida F1. Recovery efficiency of fixed cells over the course of the entire in situ detection procedure was approximately 81% for both P. putida F1 and AC10R cells. It appeared that lysozyme treatment and PCR thermal cycling were the steps responsible for most of observed cell loss. Bacterial cells expressing the todC1 gene could be discriminated from negative control cells of the same size based on flow cytometrically-measured fluorescence and forward angle light scatter. According to flow cytometric analysis, the fluorescence intensity of positive cells was 4-5 times brighter than that of negative cells. The combination of flow cytometry and a prokaryotic in situ reverse transcription-PCR (RT-PCR) approach make possible the rapid detection and enumeration of functional (based on mRNA) populations of microbial cells.
Chen
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F
Department of Marine Sciences, University of Georgia, Athens, GA 30602-2206, USA.
Binder
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B
Hodson
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RE
D005434:Flow Cytometry; D020869:Gene Expression Profiling; D017404:In Situ Hybridization, Fluorescence; D008856:Microscopy, Fluorescence; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics*; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D012329:RNA, Bacterial; D012333:RNA, Messenger; D010105:Oxygenases; C023172:toluene dioxygenase