Quantifying RNA and DNA in planktonic organisms with SYBR Green II and nucleases. Part A. Optimisation of the assay

摘要: Assay protocols for RNA and DNA in crude plankton extracts using the fluorochrome SYBR Green II are developed here. The method is based on fluorescence 3 aliquots: first measures after digestion; second third residual digestion of both RNA. This measurement critical accurate calculations nucleic acids. Optimisation assay conditions described: concentration, buffer composition, stability, temperature duration nuclease incubation. In optimised procedure, assays performed 5 mM Tris (containing 0.9 CaCl2·2H2O MgCl2·6H2O, pH 8.0); DNase RNase incubations conducted at 37°C 20 min; added to all a final concentration 3.5x10-4 readings done within 10-60 min period following addition. study evidenced importance digestion, which especially taken into account calculation acid concentrations. Finally, variability fluorescent response different standards examined; from tests, rRNA calf liver thymus standards. accompanying paper (Berdalet et al., 2005) describes development extraction protocol, as well application measuring RNA/DNA ratios natural samples, comparison with ethidium bromide methods.