Determination of Rna and Dna Concentrations in Natural Plankton Samples Using Thiazole Orange in Combination with Dnase and Rnase DIGESTIONS1 (original) (raw)
Related papers
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.
Contents lists available at SciVerse ScienceDirect Journal of Chromatography A
A residue analysis method for the simultaneous estimation of 349 pesticides, 11 PCBs and 15 PAHs extracted from grape, pomegranate, okra, tomato and onion matrices, was established by using a gas chromatograph coupled to an electron impact ionization triple quadrupole mass spectrometer (GC-EI-MS/MS). The samples were extracted by ethyl acetate and cleaned by dispersive solid phase extraction with PSA and/or GCB/C 18 by the methods reported earlier. The GC-EI-MS/MS parameters were optimized for analysis of all the 375 compounds within a 40 min run time with limit of quantification for most of the compounds at <10 g/L, which is well below their respective European Union-Maximum Residue Levels. The coefficient of determination (r 2 ) was >0.99 within the calibration linearity range of <5-250 ng/mL for compounds with LOQs < 5 ng/mL. While for the compounds with LOQs within 5-10 g/kg, the lowest calibration level was 5 and 10 g/kg as applicable. The recoveries at 10, 25 and 50 ng/mL were within 70-110% (n = 6) with associated RSDs < 20% indicating satisfactory precision. The information generated from the single laboratory validation was further utilized for building a semi-quantitative approach. The accuracies in quantification obtained via individual calibration standards vis-à-vis semiquantification approach were comparable. For incurred samples, the concentrations estimated by the semi-quantification approach were within ±10% of the values obtained by direct quantification. This approach complements the existing GC-EI-MS/MS methods by offering targeted screening and quantification capabilities.
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.
On the recalcitrant use of Arnon's method for chlorophyll determination
The New phytologist, 2017
On the recalcitrant use of Arnon's method for chlorophyll determination Some of the most widely utilized measurements in plant science research, such as total protein content or chlorophyll (chl) determination, are both straightforward and indispensable. The accuracy of the techniques employed for the quantification of these compounds is an important part of laboratory work, which will compromise the further determinations and even the conclusions of many of the articles published now and in the future. For the topic of this Letter, chl determination, a complete set of methodologies, from remote sensing to chromatographic analyses, are available (Blackburn & Ferwerda, 2008; Cortazar et al., 2015; Junker & Ensminger, 2016). Among this broad array of methodologies, one of the most (if not the most) successful protocol for the simultaneous quantification of chla and b is the one described by Arnon in the middle of last century (Arnon, 1949). Evidence of its success is the fact that it has received almost 14 000 citations up to now and it has been in the top 100 most cited papers of all time (Garfield, 1990) for decades, a position only recently lost (it ranks 139 th , July 2017). Even so, Arnon's paper is frequently cited even now (actually, it is one of the four most-cited papers ever in the area of Plant Science, according to the Web of Sciences, 2017). In fact, the highest number of citations was recorded in 2014, 2015 and 2016 (Fig. 1). Interestingly, chl determination was not the main achievement of this paper, which focused on copper metabolism in chloroplasts. However, the equations proposed for chl measurement became the reason for its success. In fact, his previous Nature paper on copper (Arnon, 1948) has only received 15 citations since its publication. Despite their great success in numeric terms and in spite of the fact that they enjoy such a huge following, Arnon's equations are based on old and inaccurate molar extinction coefficients calculated for 80% acetone as solvent.
Determination of< sup> 18 O concentrations in musamples of biological fluids
1990
Determination of 180 by prompt nuclear reaction analysis: application for measurement of microsamples. J. Appl. Physiol. 63(3): 1296-1302, 1987.-A method is described for the routine determination of 180 concentrations in microsamples of biological fluids. The method utilizes the prompt nuclear reaction 180(p,&15N, and 846-keV protons fromOa 3-MeV Van de Graaff Accelerator are focused on -2,000-A-thick Taz05 targets prepared by anodic oxidation from 50-~1 samples of water distilled from blood or other biological fluids. The broad cross section of the resonance peak for this nuclear reaction (47 keV) ensures high yields, especially at small reaction angles, and the high-energy a! particles produced by the reaction (4 MeV) are readily separated from scattered protons by the use of an aluminized Mylar foil of suitable thickness. Background levels of 180 (0.204 atom%) can be detected with run times of -5-8 min, and the sensitivity of the method is of the order of 0.05 atom%. Experimental error due to sample preparation was found to be 1.7%, and counting errors were close to theoretical limits so that total error was of the order of 2.5%. Duplicate samples were analyzed by use of the 180(p,~,)15N reaction at Lucas Heights, Australia, and the "O(p,n)18F reaction by the method of Wood et al. (Anal. Chem. 47: 646-650,1975) at the University of California, Los Angeles, and the agreement was excellent (y = 1.0123x -0.0123, r = 0.991, P < 0.001). The theoretical limitations and the general applicability of the method in biological studies designed to estimate the rate of metabolism of free-ranging animals are discussed.
The Fifth SeaWiFS HPLC Analysis Round-Robin Experiment (SeaHARRE -5)
The focus of this study was the estimation of uncertainties in quantitating a diverse set of chlorophyll and carotenoid pigment concentrations for a variety of HPLC methods and related procedures used in the analysis of predominantly coastal waters. Samples were collected from two different environments, which were anticipated to have very different HPLC baselines. The first set of 12 samples were taken from the New England (United States) coastal waters around Portsmouth, New Hampshire. The second set of 12 samples were obtained in the near vicinity of the coastal waters around Hobart, Tasmania (Australia). The latter were expected to have simpler baselines than the former, because the environmental conditions of the Tasmanian watersheds were expected to be less influenced by anthropogenic sources (as established by prior analyses). The SeaHARRE -5 activity was designed to investigate the following objectives: a) estimate the uncertainties in quantitating individual pigments and higher-order variables formed from sums and ratios; b) confirm if the chlorophyll a accuracy requirements for ocean color validation activities (approximately 25%, although 15% would allow for algorithm refinement) can be met in coastal waters; c) establish the reduction in uncertainties as a result of applying QA procedures; d) show the importance of establishing a properly defined referencing system in the computation of uncertainties; e) quantify the analytical benefits of performance metrics, f) demonstrate the utility of a laboratory mix in understanding method performance, and g) determine whether or not noisy baselines elevate pigment uncertainties. All of these objectives were satisfied and the results associated with each are presented.