Semiautomated fluorometric analysis of nucleic acids in tissue homogenates

Abstract

This report describes a technique that was developed to provide an efficient and accurate estimation of RNA:DNA ratios. These ratios have been used as an instantaneous measure of recent growth of individual aquatic organisms where morphometrics are not appropriate (e.g., field-collected species) or insufficiently sensitive (e.g., small life stages or species). In this semiautomated, sensitive method, ethidium bromide fluorescence was used to quantitate total nucleic acids in crude homogenates. Individual concentrations of RNA and DNA were determined by differences in fluorescence before and after elimination of RNA by digestion with RNase. Efficiency of the procedure was enhanced using a computer-driven multiwell plate scanning system (CYTOFLUOR, Millipore Corporation1 ) to measure fluorescence at timed intervals and perform data manipulations. Routinely, detection limits of 0.1 μg DNA and 0.4 μg RNA were achieved, allowing the analysis of small, individual organisms. Fluorescence results of split samples were comparable with those obtained using a standard spectro-photometric method to quantitate nucleic acids. Coefficients of variation for replicate samples within an assay (1.6%) and for samples within replicate assays (5.6%) indicated good test reproducibility. Quantitative recoveries of nucleic acid standards spiked into tissue homogenates were generally high, averaging 91.0% for DNA and 119.0% for RNA. Factors affecting the fluorescence of ethidium bromide stained nucleic acids—e.g., nucleic acid source, crude homogenate components, and buffer composition—are discussed relative to assay performance. This method provides a rapid and reliable assessment of individual growth, an important sublethal toxicological end point, that is suitable for both laboratory and field studies. © 1994 by John Wiley & Sons, Inc..