Pernthaler Annelie, Amann Rudolf
Max-Planck-Institut für Marine Mikrobiologie, Celsiusstrabetae 1, D-28359 Bremen, Germany.
Appl Environ Microbiol. 2004 Sep;70(9):5426-33. doi: 10.1128/AEM.70.9.5426-5433.2004.
We developed for Bacteria in environmental samples a sensitive and reliable mRNA fluorescence in situ hybridization (FISH) protocol that allows for simultaneous cell identification by rRNA FISH. Samples were carbethoxylated with diethylpyrocarbonate to inactivate intracellular RNases and pretreated with lysozyme and/or proteinase K at different concentrations. Optimizing the permeabilization of each type of sample proved to be a critical step in avoiding false-negative or false-positive results. The quality of probes as well as a stringent hybridization temperature were determined with expression clones. To increase the sensitivity of mRNA FISH, long ribonucleotide probes were labeled at a high density with cis-platinum-linked digoxigenin (DIG). The hybrid was immunocytochemically detected with an anti-DIG antibody labeled with horseradish peroxidase (HRP). Subsequently, the hybridization signal was amplified by catalyzed reporter deposition with fluorochrome-labeled tyramides. p-Iodophenylboronic acid and high concentrations of NaCl substantially enhanced the deposition of tyramides and thus increased the sensitivity of our approach. After inactivation of the antibody-delivered HRP, rRNA FISH was performed by following routine protocols. To show the broad applicability of our approach, mRNA of a key enzyme of aerobic methane oxidation, particulate methane monooxygenase (subunit A), was hybridized with different types of samples: pure cultures, symbionts of a hydrothermal vent bivalve, and even sediment, one of the most difficult sample types with which to perform successful FISH. By simultaneous mRNA FISH and rRNA FISH, single cells are identified and shown to express a particular gene. Our protocol is transferable to many different types of samples with the need for only minor modifications of fixation and permeabilization procedures.
我们为环境样品中的细菌开发了一种灵敏且可靠的mRNA荧光原位杂交（FISH）方案，该方案可通过rRNA FISH同时进行细胞鉴定。样品用焦碳酸二乙酯进行乙氧羰基化处理以灭活细胞内的核糖核酸酶，并分别用不同浓度的溶菌酶和/或蛋白酶K进行预处理。事实证明，优化每种样品的通透处理是避免出现假阴性或假阳性结果的关键步骤。利用表达克隆确定探针质量以及严格的杂交温度。为提高mRNA FISH的灵敏度，长核糖核苷酸探针用顺铂连接的地高辛配基（DIG）进行高密度标记。用辣根过氧化物酶（HRP）标记的抗DIG抗体对杂交体进行免疫细胞化学检测。随后，通过用荧光染料标记的酪胺进行催化报告沉积来放大杂交信号。对碘苯硼酸和高浓度的氯化钠可显著增强酪胺的沉积，从而提高我们方法的灵敏度。在抗体提供的HRP失活后，按照常规方案进行rRNA FISH。为证明我们方法的广泛适用性，将需氧甲烷氧化关键酶颗粒性甲烷单加氧酶（亚基A）的mRNA与不同类型的样品进行杂交：纯培养物、热液喷口双壳贝类的共生体，甚至是沉积物，沉积物是最难成功进行FISH的样品类型之一。通过同时进行mRNA FISH和rRNA FISH，可鉴定单个细胞并显示其表达特定基因。我们的方案可转移至许多不同类型的样品，只需对固定和通透处理程序进行少量修改即可。
