The Development of a Rapid and Sensitive, High-Through-Put Protocol for RNA:DNA Ratio Analysis

Abstract

We present a new technique for determining RNA:DNA ratios in larval and juvenile fish that relies on the rapid homogenization of samples with salts, detergents, and proteinase K and the detection of DNA and RNA with specific fluorescent dyes. Samples are not purified before assay of nucleic acids. The technique was designed for use in 96 or 384 well plates and required only 50 μL of sample per well. The detection of RNA and DNA is linear over 500 ng and is sensitive to a nucleic acid content as little as that within 50 μg of tissue. The new technique was used to characterize the RNA:DNA ratio in the white muscle of juvenile mummichogs Fundulus heteroclitus and larval winter flounder Pleuronectes americanus. This technique was compared with other techniques that use fluorescent dyes to determine the RNA:DNA ratio in fish samples.