New double staining technique for the RNA and DNA measurement in marine phytoplankton (original) (raw)
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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.
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. ...
Scientia Marina, 2005
Assay protocols for RNA and DNA in crude plankton extracts using the fluorochrome SYBR Green II are developed here. The method is based on the fluorescence in 3 aliquots: the first measures RNA after DNA digestion; the second measures DNA after RNA digestion; and the third measures residual fluorescence after digestion of both DNA and RNA. This residual fluorescence measurement is critical for accurate calculations of the nucleic acids. Optimisation of the assay conditions are described: fluorochrome concentration, buffer composition, fluorescence stability, temperature and duration of nuclease incubation. In the optimised procedure, the assays are performed in 5 mM Tris buffer (containing 0.9 mM CaCl 2 •2H 2 O and 0.9 mM MgCl 2 •6H 2 O, pH 8.0); DNase and RNase incubations are conducted at 37ºC for 20 min; the fluorochrome is added to all assays at a final concentration of 3.5x10-4 and readings are done within the 10-60 min period following the SYBR Green II addition. The study evidenced the importance of the residual fluorescence after nuclease digestion, which is especially taken into account in the calculation of the nucleic acid concentrations. Finally, the variability of the fluorescent response to different RNA and DNA standards is examined; from the performed tests, calculations are based on rRNA from calf liver and DNA from calf thymus standards. The accompanying paper (Berdalet et al., 2005) describes the development of the extraction protocol, as well as the application of both protocols in measuring RNA/DNA ratios in natural plankton samples, and a comparison with ethidium bromide based methods.
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.
Development of new procedures for the isolation of phytoplankton DNA from fixed samples
Journal of Applied Phycology, 2005
Phytoplankton samples collected for routine monitoring programmes have traditionally been preserved with fixatives before subsequent analytical procedures such as microscope-based identification, or simply to permit transport between laboratories. In recent years, to simplify identification and enumeration, the use of DNA or RNA probes coupled with the PCR assay has progressed and now represents a routine procedure for screening cultured and field samples. However, the phytoplankton cells have often still to be treated as fixed samples.
Determination of dissolved nucleic acids in seawater by the fluorescence dye, ethidium bromide
Marine Chemistry, 1992
A new method for the determination of dissolved double-stranded deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in seawater was developed, evaluated and used to study the fates of these nucleic acids in marine ecosystems. These nucleic acids, which were pre-concentrated on a hydroxyapatite column, were determined fluorometrically by the use of ethidium bromide dye, which binds specifically to the double-stranded polynucleotide. No dissolved organic matter coexisting in the preconcentrated sample solution interfered in the analysis of DNA and RNA. Column recoveries of DNA and RNA in a sample volume of up to 11 were 93% and 97%, respectively, and 90% of both at 51. The detection limits of DNA and RNA concentrated from a 5 1 sample by this fluorometric method were 0.6 and 1.1/tg 1-1, respectively. The concentration of dissolved nucleic acids in the waters from Tokyo Bay and Sagami Bay showed great variation in space and time. DNA ranged from 1 to 32 #g 1-1, and RNA from below the detection limit to 34/tg 1-I. The total amount of phosphorus in nucleic acids was an important fraction (12.9 + 8.2%) of the dissolved organic phosphorus (DOP) and showed a good correlation with DOP.
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.
Journal of Phycology, 1996
'4 juorometric technique, h(isec1 O H the co,nbinatioji of R,Vase a n d D S a s e incubatioji ic'ith the u w of thiazole orange (RSasei D-Yase method), x,as inilestigated to determine D.VA a n d R-YA concentrations i,i marine plankt o x Tests uiere perjortned to optittiiz both RA\\nse and DSnse assaj conditions. The R.Ycisr c l m j should be conducted at 3 i O C f o r 20 min ;cmith 0.5 pg.viL-' of D S a s efree RSase. An incubation nt 23" C f o r 20 mi)z u i t h 10 units. mL-' of R-Yase-free DAYitse xere the optimal conditions required f o r DlYA4 cligrstioji t)j D.Yase. T h e detection limits i n terms of viiniut u i n bioviassfor reliable measurevteuts of D1\ ;4 and R.Y.4 ~C ' C~C 7.5 a n d 10 pg of protein. (OIL nssaj)l , respectively. RNA and DNA concen frations were esfimated in oligotrolhic ulater samples usilig the R.Yasei D. \' ase a n d other azlailable methods (e.g. a cloiiblejlrtororh rome method). T h e different techniques proilided simila r DL\:4 estimations. Houleiler, the RSnse i DSase method proilided the highest sensitiilitj a n d (I loz, mriabilitj for the estimation of RLYL4.
Applied and Environmental Microbiology, 2002
The aim of this study was to develop a protocol for the simultaneous extraction from bacterioplankton of RNA and DNA suitable for quantitative molecular analysis. By using a combined mechanical and chemical extraction method, the highest RNA and DNA yield was obtained with sodium lauryl sarcosinate-phenol or DivoLab-phenol as the extraction mix. The efficiency of extraction of nucleic acids was comparatively high and varied only moderately in gram-negative bacterial isolates and bacterioplankton (RNA, 52 to 66%; DNA, 43 to 61%); significant amounts of nucleic acids were also obtained for a gram-positive bacterial isolate (RNA, 20 to 30%; DNA, 20 to 25%). Reverse transcription-PCR and PCR amplification products of fragments of 16S rRNA and its genes were obtained from all isolates and communities, indicating that the extracted nucleic acids were intact and pure enough for community structure analyses. By using single-strand conformation polymorphism of fragments of 16S rRNA and its g...