TY - JOUR
T1 - 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)
AU - Kubota, Kengo
AU - Ohashi, Akiyoshi
AU - Imachi, Hiroyuki
AU - Harada, Hideki
N1 - Funding Information:
Madan Tandukar at Nagaoka University of Technology is acknowledged for reading this manuscript. This study was supported financially by research grants from the New Energy and Industrial Technology Development Organization (NEDO), Japan and the Ministry of Education, Culture, Sports, Science and Technology, Japan.
PY - 2006/9
Y1 - 2006/9
N2 - 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.
AB - 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.
KW - Fluorescence in situ hybridization (FISH)
KW - Methanococcus vannielii
KW - Methanogen
KW - Methyl coenzyme M reductase (mcr)
KW - mRNA
KW - Tyramide signal amplification (TSA)
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U2 - 10.1016/j.mimet.2006.02.002
DO - 10.1016/j.mimet.2006.02.002
M3 - Article
C2 - 16545875
AN - SCOPUS:33745946807
SN - 0167-7012
VL - 66
SP - 521
EP - 528
JO - Journal of Microbiological Methods
JF - Journal of Microbiological Methods
IS - 3
ER -