Visualization of mcr mRNA in a methanogen by fluorescence in situ hybridization with an oligonucleotide probe and two-pass tyramide signal amplification (two-pass TSA-FISH).
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.
Kubota
Kengo
K
Department of Environmental Systems Engineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan.
Ohashi
Akiyoshi
A
Imachi
Hiroyuki
H
Harada
Hideki
H
D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D017017:Methanococcus / Q000201:enzymology* / Q000235:genetics; D008856:Microscopy, Fluorescence; D015345:Oligonucleotide Probes; D010088:Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D010088:Oxidoreductases; D006735:Horseradish Peroxidase; C021572:methyl coenzyme M reductase