A High Through-Put Protocol for Quantifying Nucleic Acids in Individual Microcrustaceans Using New Generation RNA and DNA Specific Dyes

Abstract

Quantifying growth rate in microcrustacea is important to plankton biologists when determining primary productivity. RNA:DNA ratios have been widely used as an indicator of instantaneous growth rate in microcrustaceans in nutritional and environmental studies. It is important when measuring nucleic acids in these small organisms that it is done efficiently and with high sensitivity, so that small differences in nucleic acid content can be accurately detected. A technique is presented for the accurate, high-throughput determination of RNA and DNA content in individual microcrustacea, based on two new generation fluorescent dyes that bind preferentially to either RNA (Quant-iT™ RNA) or DNA (Quant-iT™ DNA). Contaminant interference (either from detergents or enzymes used in the chemical cell lysis process, or cell based contaminants such as proteins) can significantly affect fluorescence readings of these dyes. Through the development of our protocol, we show that proteinase K was an ineffective cell lysis method, whereas sarcosyl efficiently lysed cells and could be used when its final assay concentration was diluted to avoid interference with dye fluorescence. Organic solvents were also utilized to remove cell based contaminants that caused an interference with the Quant-iT™ DNA dye fluorescence. Through the use of specific RNA and DNA dyes and by achieving low levels of technical variation, this study developed a rapid and reliable method for nucleic acid extraction and quantification in Artemia.