Dell'Anno A, Fabiano M, Duineveld GCA, Kok A, Danovaro R
Faculty of Science, Marine Science, University of Ancona, Ancona, Italy.
Appl Environ Microbiol. 1998 Sep;64(9):3238-45. doi: 10.1128/AEM.64.9.3238-3245.1998.
In this study, we compared three methods for extraction and quantification of RNA and DNA from marine sediments: (i) a spectrophotometric method using the diphenylamine assay; (ii) a fluorometric method utilizing selective fluorochromes (thiazole orange for total nucleic acids and Hoechst 33258 for DNA); and (iii) a high-pressure liquid chromatography (HPLC) method which uses a specific column to separate RNA and DNA and UV absorption of the nucleic acids for quantification. Sediment samples were collected in the oligotrophic Cretan Sea (eastern Mediterranean, from 40 to 1,540 m in depth) and compared to the distribution and composition of the benthic microbial assemblages (i.e., bacteria and microprotozoa). DNA concentrations measured spectrophotometrically and by HPLC were not significantly different, while fluorometric yields were significantly lower. Such differences appear mainly due to fact that the stain-DNA complex is strongly dependent on the DNA composition and structure. RNA concentrations determined by the three methods displayed some differences; fluorometric and spectrophotometric methods obtain RNA concentration by difference and therefore may be biased by DNA estimates. By contrast, the HPLC method provides independent assessments of RNA and DNA concentrations. We tentatively estimated the contribution of the detrital DNA to the total DNA pools in two ways. The two calculations provided quite similar results indicating that the majority of the DNA pool in the deep-sea sediments was detrital. Microbial RNA generally accounted for almost the entire sedimentary RNA pools below 100-m depth. RNA concentrations were found to decrease along the Cretan shelf and slope. The RNA/DNA ratio calculated by using fluorometric DNA concentrations was significantly correlated with values of sediment community oxygen consumption only below 100-m depth (dominated by the microbial biomass). These data suggest that the RNA/DNA ratio, based on fluorometric estimates of DNA, can be used as an indicator of benthic metabolic activity, but only when metazoan contribution to the microbial DNA is negligible.
在本研究中，我们比较了从海洋沉积物中提取和定量RNA与DNA的三种方法：（i）使用二苯胺测定法的分光光度法；（ii）利用选择性荧光染料的荧光法（噻唑橙用于总核酸，Hoechst 33258用于DNA）；以及（iii）高压液相色谱（HPLC）法，该方法使用特定的柱子分离RNA和DNA，并利用核酸的紫外吸收进行定量。沉积物样本采集于贫营养的克里特海（地中海东部，深度为40至1540米），并与底栖微生物群落（即细菌和微型原生动物）的分布和组成进行比较。通过分光光度法和HPLC测定的DNA浓度没有显著差异，而荧光法的产量则显著较低。这些差异主要是由于染色-DNA复合物强烈依赖于DNA的组成和结构。用这三种方法测定的RNA浓度存在一些差异；荧光法和分光光度法通过差值获得RNA浓度，因此可能受到DNA估计值的影响而产生偏差。相比之下，HPLC法可独立评估RNA和DNA的浓度。我们尝试用两种方法初步估计碎屑DNA对总DNA库的贡献。两种计算结果相当相似，表明深海沉积物中大部分DNA库是碎屑性的。微生物RNA通常几乎占100米深度以下整个沉积RNA库的全部。发现RNA浓度沿克里特陆架和陆坡降低。仅在100米深度以下（以微生物生物量为主），使用荧光法测定的DNA浓度计算出的RNA/DNA比值与沉积物群落耗氧量值显著相关。这些数据表明，基于荧光法估计的DNA得出的RNA/DNA比值可作为底栖代谢活动的指标，但前提是后生动物对微生物DNA的贡献可忽略不计。
