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.