Determination of dissolved nucleic acids in seawater by the fluorescence dye, ethidium bromide (original) (raw)
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Applied and environmental microbiology, 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. ...
Applied and Environmental Microbiology
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.
The measurement and distribution of dissolved nucleic acids in aquatic environments
Limnology and Oceanography, 1989
Nucleic acids (DNA and RNA) are ubiquitous components of the dissolved organic matter (DOM) pool of all oceanic, neritic, estuarine, and freshwater habitats studied to date. A new method for the quantitative determination ofdissolved nucleic acids (DNA and RNA) in water and scdimcnt samples was developed, evaluated, and utilized in a study of various marine and freshwater ecosystems. Under appropriate reaction conditions, dissolved DNA (D-DNA) and dissolved RNA (D-RNA) are efficiently removed from solution with the addition of cctyltrimethylammonium bromide (CTAB) and subsequent formation of insoluble CTA-nucleic acid salts. The insoluble salts are collected, by filtration, onto glass-fiber filters and analyzed for DNA and RNA with fluorometric and calorimetric procedures, respectively. The pcrformancc of this CTAB method is simple, reliable, and reproducible for measuring dissolved nucleic acids in natural aquatic environments. For the ecosystems investigated hcrcin, D-DNA and D-RNA concentrations ranged from 0.56 to 88 pg liter-' and 4.03 to 871 pg liter I; the ratio of D-RNA to D-DNA ranged from 4.1 to 11.5.
New double staining technique for the RNA and DNA measurement in marine phytoplankton
1991
The use of RNA: DNA ratios a s a biochemical indicator is hampered by the lack of simple and reliable methods for routine work. The technique presented here approaches this problem by using 2 fluorochromes: Hoechst 33258, which specifically reacts ~l t h DNA, a n d Thiazole Orange, which allows total nucleic acid estimation. Samples dre honlogenized in ~ris-Ca'+ buffer which is also used in the fluorometric analysis. Subsequently, these fluorochromes are added to different subsamples of the same nucleic arid extract. Several extraction and analysis techniques were tested and an optimized procedure is described The technique 1s simple and more sensitive than previously described methods for the determination of RNA and DNA In phytoplankton.
New double-staining technique for RNA and DNA measurement in marine phytoplankton
Marine Ecology Progress Series, 1991
The use of RNA: DNA ratios a s a biochemical indicator is hampered by the lack of simple and reliable methods for routine work. The technique presented here approaches this problem by using 2 fluorochromes: Hoechst 33258, which specifically reacts ~l t h DNA, a n d Thiazole Orange, which allows total nucleic acid estimation. Samples dre honlogenized in ~ris-Ca'+ buffer which is also used in the fluorometric analysis. Subsequently, these fluorochromes are added to different subsamples of the same nucleic arid extract. Several extraction and analysis techniques were tested and an optimized procedure is described The technique 1s simple and more sensitive than previously described methods for the determination of RNA and DNA In phytoplankton.
Marine Ecology Progress Series
The use of RNA: DNA ratios a s a biochemical indicator is hampered by the lack of simple and reliable methods for routine work. The technique presented here approaches this problem by using 2 fluorochromes: Hoechst 33258, which specifically reacts ~l t h DNA, a n d Thiazole Orange, which allows total nucleic acid estimation. Samples dre honlogenized in ~ris-Ca'+ buffer which is also used in the fluorometric analysis. Subsequently, these fluorochromes are added to different subsamples of the same nucleic arid extract. Several extraction and analysis techniques were tested and an optimized procedure is described The technique 1s simple and more sensitive than previously described methods for the determination of RNA and DNA In phytoplankton.
A novel method for the measurement of dissolved deoxyribonucleic acid in seawater
A novel method was developed for the quantification of dissolved deoxyribonucleic acid (D-DNA) in seawater. This method includes addition of tetrasodium ethylenediamine tetraacetic acid (tetrasodium EDTA) to 0.22 µmfiltered seawater, concentration of > 10 kDa material in the filtrate with a Centricon centrifugal concentration unit, and quantification of the concentrated D-DNA with the fluorescent double-stranded DNA stain SYBR Green I. This method requires less than 15 mL of seawater per sample even in oligotrophic environments, and samples can be analyzed in approximately 3 h. The recovery of D-DNA with this method is 75% to 85% and can be determined for each sample by measuring recovery of 35 S-labeled DNA added at trace amounts. This method can be used to quantify D-DNA concentrations as low as 0.01 ng mL -1 with high precision (standard deviation < 5% of the mean). Deoxyribonuclease (DNase) treatment of samples and virus enumeration can be used in conjunction with this method to determine the three major components of D-DNA: free or enzymatically hydrolyzable DNA (ehD-DNA), DNA within viruses, and uncharacterized bound DNA.
Comments on the determination of nucleic acids in natural waters by the CTAB-DABA-orcinol method
Science of The Total Environment, 1996
Karl and Bailiff (1989) presented the CTAB-DABA-orcinol method for the determination of dissolved DNA (D-DNA) and RNA (D-RNA) in natural waters. Here we demonstrate that, by this method, not only RNA but also heteropolysaccharides containing pentose sugars are precipitated and measured. RNase treatment resulted in only a slight reduction of the measured D-RNA concentrations in seawater. These findings indicate that the CTAB-orcinol method is not specific for measuring D-RNA. We improved the accuracy of the CTAB-DABA assay for determining D-DNA in seawater and we further show that viral DNA is probably also measured by the CTAB-DABA method.
RNA:DNA Ratio and Other Nucleic Acid Derived Indices in Marine Ecology
International Journal of Molecular Sciences, 2008
Some of most used indicators in marine ecology are nucleic acid-derived indices. They can be divided by target levels in three groups: 1) at the organism level as ecophysiologic indicators, indicators such as RNA:DNA ratios, DNA:dry weight and RNA:protein, 2) at the population level, indicators such as growth rate, starvation incidence or fisheries impact indicators, and 3) at the community level, indicators such as trophic interactions, exergy indices and prey identification. The nucleic acids derived indices, especially RNA:DNA ratio, have been applied with success as indicators of nutritional condition, well been and growth in marine organisms. They are also useful as indicators of natural or anthropogenic impacts in marine population and communities, such as upwelling or dredge fisheries, respectively. They can help in understanding important issues of marine ecology such as trophic interactions in marine environment, fish and invertebrate recruitment failure and biodiversity changes, without laborious work of counting, measuring and identification of small marine organisms. Besides the objective of integrate nucleic acid derived indices across levels of organization, the paper will also include a general characterization of most used nucleic acid derived indices in marine ecology and also advantages and limitations of them. We can conclude that using indicators, such RNA:DNA ratios and other nucleic acids derived indices concomitantly with organism and ecosystems measures of responses to climate change (distribution, abundance, activity, metabolic rate, survival) will allow for the development of more rigorous and realistic predictions of the effects of anthropogenic climate change on marine systems.