Abstract
Two-pass tyramide signal amplification-fluorescence in situ hybridization (two-pass TSA-FISH) with a horseradish peroxidase (HRP)-labeled oligonucleotide probe was applied to detect prokaryotic mRNA. In this study, mRNA of a key enzyme for methanogenesis, methyl coenzyme M reductase (mcr), in Methanococcus vannielii was targeted. Applicability of mRNA-targeted probes to in situ hybridization was verified by Clone-FISH. It was observed that sensitivity of two-pass TSA-FISH was significantly higher than that of TSA-FISH, which was further increased by the addition of dextran sulphate in TSA working solution. Signals from two-pass TSA-FISH were more reliable compared to the weak, spotty signals yielded by TSA-FISH.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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DNA, Bacterial / chemistry
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DNA, Bacterial / genetics
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Fluorescent Dyes / chemistry
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Horseradish Peroxidase / chemistry
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In Situ Hybridization, Fluorescence / methods*
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Methanococcus / enzymology*
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Methanococcus / genetics
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Microscopy, Fluorescence
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Oligonucleotide Probes
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Oxidoreductases / genetics*
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Oxidoreductases / isolation & purification
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Polymerase Chain Reaction
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RNA, Messenger / analysis*
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RNA, Messenger / genetics
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RNA, Ribosomal, 16S / chemistry
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RNA, Ribosomal, 16S / genetics
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Sensitivity and Specificity
Substances
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DNA, Bacterial
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Fluorescent Dyes
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Oligonucleotide Probes
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RNA, Messenger
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RNA, Ribosomal, 16S
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Oxidoreductases
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Horseradish Peroxidase
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methyl coenzyme M reductase