DETERMINATION OF RNA AND DNA CONCENTRATIONS IN NATURAL PLANKTON SAMPLES USING THIAZOLE ORANGE IN COMBINATION WITH DNASE AND RNASE DIGESTIONS¹

Abstract

A fluorometric technique, based on the combination of RNase and DNase incubation with the use of thiazole orange (RNase/DNase method), was investigated to determine DNA and RNA concentrations in marine plankton. Tests were performed to optimize both RNase and DNase assay conditions. The RNase assay should be conducted at 37° C for 20 min with 0.5 μg·mL−1 of DNase‐free RNase. An incubation at 25° C for 20 min with 10 units ·mL‐1 of RNase‐free DNase were the optimal conditions required for DNA digestion by DNase. The detection limits in terms of minimum biomass for reliable measurements of DNA and RNA were 7.5 and 10 μg of protein · (mL assay)−1, respectively. RNA and DNA concentration were estimated in oligotrophic water samples using the RNase/DNase and other available methods (e.g. a double fluorochrome method). The different techniques provided similar DNA estimations. However, the RNase/DNase method provided the highest sensitivity and a low variability for the estimation of RNA.

Quantifying RNA and DNA in planktonic organisms with SYBR Green II and nucleases. Part B. Quantification in natural samples

“…This level of sensitivity is ca. 20 times higher than our previous method, based on thiazole orange (Fara et al, 1996), which we used for microplankton samples. For larvae, we fixed a minimum biomass requirement at 3 µg DW (ml assay) -1 (ca.…”

“…Our laboratory had been measuring RNA and DNA of microplankton or copepod samples collected on GF/F glass fibre filters (Berdalet and Dortch, 1991;Fara et al, 1996;Saiz et al, 1998). Extraction was conducted by simply grinding the filters in a Tris buffer.…”

Rates of growth and microbial grazing mortality of phytoplankton in a recent artificial lake

“…Proteins were analyzed according to Lowry et al (1951) using bovine serum albumin as a standard. Nucleic acids were determined fluorometrically by the enzymatic method of Fara et al (1996). Briefly, Whatman GF/F filters with collected organisms onto were ground in Tris buffer (5 mg ml -1 ) and 1 ml aliquot of the supernatant was spiked with 0.5 µg ml -1 of DNase for RNA measurements or with 10 units ml -1 of RNase for DNA quantification, and incubated for 20 min at 37°C.…”

“…Briefly, Whatman GF/F filters with collected organisms onto were ground in Tris buffer (5 mg ml -1 ) and 1 ml aliquot of the supernatant was spiked with 0.5 µg ml -1 of DNase for RNA measurements or with 10 units ml -1 of RNase for DNA quantification, and incubated for 20 min at 37°C. After cooling at room temperature, 0.55 ml of Thiazole Orange (10 µg ml -1 , final concentration) was added to each sample and the fluorescence determined at 511 and 533 nm for the calculation of nucleic acid concentrations (Fara et al 1996). We were interested in particulate protein and nucleic acids because nitrogen is a component of both protein and nucleic acids and phosphorus is essential in the nucleic acid skeleton (Nelson & Cox 2000).…”

“…Furthermore, RNA is directly implicated in protein synthesis and has been considered as an indicator of growth in several groups of microorganisms (Dortch et al 1983). One could thus expect that the shortage of N or P in the Sep Reservoir would differently affect the internal amounts of particulate protein and nucleic acids and thus the growth rates of microbial communities (De Madariaga & Joint 1992, Berdalet et al 1996.…”