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

Abstract

Extraction procedures for RNA and DNA in crude extracts of natural microplankton samples are developed. The methodology is compatible with the fluorometric assay of RNA and DNA developed by Berdalet et al. (2005). Mechanical cell disruption using a manual tissue grinder is combined with chemical extraction using 0.5% sarcosine and 0.5 mM EDTA in 5 mM Tris, pH 8.0, to release the nucleic acid. The new extraction and assay procedure is used to estimate the RNA/DNA ratios of fish larvae and natural microplankton communities maintained in the laboratory under different nutritional conditions. In the two experiments, the RNA/DNA ratios were related to the nutrient availability of the organisms. The level of sensitivity of the method is experimentally set at ca. 40 ng of RNA and 10 ng DNA in the 1 ml assay, which corresponds to a minimum biomass requirement of ca. 400 ng ml^-1 protein or 3 µg ml^-1 dry weight. The precision, estimated at different steps of the procedure, had an overall coefficient of variation of ?10%. The new approach provides RNA/DNA ratios comparable with those obtained with ethidium bromide.