An evaluation of fluorescence technigues for measuring DNA and RNA in marine microorganisms

Abstract

The DNA-specific fluorochrome Hoechst 33258 (H33258) and the DNA- and RNA-sensitive fluorochrome ethidium homodimer (EthDi) were used to detect ng ml-1 concentrations of nucleic acids in marine microorganisms. These fluorochromes were 4 to 5 times more sensitive to DNA than ethidium bromide, and their use significantly reduced the amount of sample necessary to measure nucleic acids in seawater. Both fluorochromes responded in a linear fashion to standard DNA, but the response of EthDi to RNA was non-linear for the majority of RNA types tested. Chlorophyll a quenched fluorescence of both fluorochromes, especially of EthDi, at concentrations that could be encountered in sample homogenates. Two techniques were used to measure DNA and RNA on cultures of marine bacteria and phytoplankton; H33258 and EthDi together (double fluorochrome technique), and EthDi with and without RNase (RNase digestion method). In general, DNA and RNA were measured equally well using either technique; discrepancies between techniques were attributed to EthDi quenching or the specificity of H33258 for DNA rich in dA-dT base pairs. The double fluorochrome technique was used to determine depth profiles of DNA and RNA/DNA in size-fractionated natural seawater. These properties showed significant changes between the surface and 100 m and may be related to changes in biomass or metabolic activity. Although consideration must be given to the RNA standard used and the presence of naturally occuring pigments, the combination of H33258 and EthDi can provide a rapid and sensitive measure of nucleic acids in seawater without the use of nucleases.