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)
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)
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DOI：
10.1016/j.mimet.2006.02.002
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发表时间：
2006-09-01
影响因子：
2.2
通讯作者：
Harada, Hideki
中科院分区：
文献类型：
--
作者：
Kubota, Kengo;Ohashi, Akiyoshi;Harada, Hideki
采用双通酪酰胺信号扩增-荧光原位杂交（Two-pass TSA-FISH）和辣根过氧化物酶（HRP）标记的寡核苷酸探针检测原核mRNA。本研究以vanielii甲烷球菌中甲烷生成的关键酶甲基辅酶M还原酶（mer）的mRNA为目标。克隆- fish验证了mrna靶向探针在原位杂交中的适用性。结果表明，两道TSA- fish的灵敏度明显高于TSA- fish，在TSA工作液中加入葡聚糖硫酸盐进一步提高了TSA- fish的灵敏度。与TSA-FISH产生的微弱、不均匀的信号相比，双通道TSA-FISH信号更可靠。(c) 2006 Elsevier b.v.版权所有
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 (mer), 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. (c) 2006 Elsevier B.V All rights reserved.