Simultaneous fluorescence in situ hybridization of mRNA and rRNA for the detection of gene expression in environmental microbes

被引:26
作者
Pernthaler, A
Pernthaler, J
机构
来源
ENVIRONMENTAL MICROBIOLOGY | 2005年 / 397卷
关键词
D O I
10.1016/S0076-6879(05)97021-3
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
A protocol is presented for the detection of gene expression in environmental microorganisms by means of fluorescence in situ hybridization (FISH). Messenger RNA (mRNA) is hybridized with digoxigenin (DIG)or fluorescein (FLUOS)-labeled ribonucleotide probes. Subsequently the hybrid is detected immunochemically with a horseradish peroxidase (HRP)-labeled antibody and tyramide signal amplification (catalyzed reporter deposition, CARD). After mRNA FISH, microorganisms can be identified by rRNA FISH with oligonucleotide probes labeled either with a fluorochrome or with HRP. Sample preparation and cell permeabilization strategies for various microbial cell types are discussed. The synthesis of DIG- and FLUOS-labeled probes, as well as custom labeling of tyramides with different fluorochromes, is described. As a case study, we describe in detail mRNA FISH of the particulate methane-monooxygenase, subunit A (pmoA) in endosymbiotic bacteria from tissue sections of a marine mollusc. PmoA is used as a marker gene for methanotrophy.
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页码:352 / +
页数:21
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