Nucleic Acid (DNA, RNA) Quantification and RNA/DNA Ratio Determination in Marine Sediments: Comparison of Spectrophotometric, Fluorometric, and HighPerformance Liquid Chromatography Methods and Estimation of Detrital DNA

Abstract

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.

Metabolically active microbial communities in uranium-contaminated subsurface sediments

“…However, previous studies have shown that an increased proportion of SSU rRNA molecules to SSU rRNA genes per cell can be observed in highly metabolically active cells (Nomura et al, 1984). Therefore, highly active taxa with low cell counts may be underrepresented or not detected in DNA-derived clone libraries because SSU rRNA gene targets are at or below detection limits (Dell'Anno et al, 1998;Nogales et al, 2001;Mills et al, 2005). The opposite would be observed if the cells were numerous but had low or no metabolic activity.…”

Sulfate‐reducing bacteria in marine sediment (Aarhus Bay, Denmark): abundance and diversity related to geochemical zonation

“…Prior to nucleic acid extraction, one set of sediment samples were exposed to EMA. Since extracellular DNA may adsorb to sediment particles and survive for many years as intact DNA (Dell'Anno et al, 1998;Demanèche et al, 2001), an EMA pre-treatment of sediment samples was performed. Like ethidium bromide, EMA is a DNA/RNA intercalating agent that can only enter bacterial cells with damaged walls.…”

Archaeal communities associated with shallow to deep subseafloor sediments of the New Caledonia Basin

“…depth (RNA18T150, RNA19T150, DNA18T150 and DNA19T150). The ratios between the 16S rRNA gene and 16S rRNA per cell have been reported to be proportional to the metabolic activity of the cells (Dell'Anno et al, 1998), the rRNA content per cell increasing with metabolic activity. As the MG-1 lineage was detected in RNA18T150 and RNA19T150 libraries, whereas absent in DNA18T150 and DNA19T150 libraries, we suggest that the sediments at 1.5 mbsf could have low cell concentrations of very active MG-1 lineage.…”