Pernthaler Annelie, Pernthaler Jakob
Department of Molecular Ecology, Max Planck Institute for Marine Microbiology, Bremen, Germany.
Methods Enzymol. 2005;397:352-71. doi: 10.1016/S0076-6879(05)97021-3.
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.
本文介绍了一种通过荧光原位杂交（FISH）检测环境微生物中基因表达的方法。信使核糖核酸（mRNA）与地高辛（DIG）或荧光素（FLUOS）标记的核糖核苷酸探针杂交。随后，通过辣根过氧化物酶（HRP）标记的抗体和酪胺信号放大（催化报告沉积，CARD）对杂交体进行免疫化学检测。在mRNA FISH之后，可以使用荧光染料或HRP标记的寡核苷酸探针通过rRNA FISH鉴定微生物。讨论了针对各种微生物细胞类型的样品制备和细胞通透策略。描述了DIG和FLUOS标记探针的合成，以及用不同荧光染料对酪胺进行定制标记。作为一个案例研究，我们详细描述了来自海洋软体动物组织切片的内共生细菌中颗粒甲烷单加氧酶A亚基（pmoA）的mRNA FISH。PmoA用作甲烷营养的标记基因。
