Determination of RNA and DNA concentrations in natural plankton samples using thiazole orange in combination with DNase and RNase digestions

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 degrees C for 20 min with 0.5 mu g . mL(-1) of DNase-free RNase. An incubation at 25 degrees 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 mu g of protein .(mL assay)(-1), respectively. RNA and DNA concentrations 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.