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

Abstract

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 CaCl2·2H2O and 0.9 mM MgCl2·6H2O, pH 8.0); DNase and RNase incubations are conducted at 37°C for 20 min; the fluorochrome is added to all assays at a final concentration of 3.5×10-4 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.