A simple high performance liquid chromatography method for the measurement of nucleobases and the RNA and DNA content of cellular material
Limnol. Oceanogr. Methods 10:608-616 (2012) | DOI: 10.4319/lom.2012.10.608
ABSTRACT: Current methods are not generally suitable for the quantitative analysis of nucleobases in cellular material and more complex matrices that are often encountered with environmental samples. Simple and reliable quantitative methods for nucleobase analysis in purified ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), microorganisms, and environmental samples are presented. Novel microwave-assisted vapor phase and liquid phase hydrolyses with HCl (6 mol L-1) were developed for the analysis of nucleobases in soluble and particulate samples, respectively. Optimal conditions for the release of purines (130°C, 10 min) and pyrimidines (160°C, 40 min) from DNA and RNA were applicable to both the vapor phase and liquid phase hydrolysis. Residual hydrochloric acid in hydrolysate can be removed easily by drying under a stream of nitrogen gas. Nucleobases were separated and quantified using high performance liquid chromatography with diode array detection (HPLC-DAD). The limit of detection for primary nucleobases ranged from 20 to 32 nmol L-1. Total hydrolysable nucleobases were analyzed in salmon testes DNA, yeast RNA, cultures of marine microalgae and cyanobacteria, and suspended particles from an estuary. The RNA and DNA contents and RNA/DNA ratios in cellular material were estimated from analyses of hydrolysable nucleobases.