Separation of high-molecular mass RNAs by high-performance liquid chromatography on hydroxyapatite

High-molecular-mass RNAs [transfer-(t-), 5S-, 18S- and 28S-RNA] in 25 mM sodium acetate buffer (pH 6.0) were separated by high-performance liquid chromatography (HPLC) on hydroxyapatite using a linear gradient (120 min-duration) from 0.03 to 0.147 M of phosphate buffer (pH 7.0) containing 0.3 M potassium chloride and 1 mM sodium azide with a slope of 2 mM/ml at a flow-rate of 0.5 ml/min. When the RNAs were dissolved in 4 M guanidine isothiocyanate-25 mM sodium acetate buffer (pH 6.0)-0.1 M beta-mercaptoethanol (4 M GIT), t-, 5S- and 18S- or 28S-RNAs but not 18S- and 28S-RNAs were separated. RNAs extracted from rat superior cervical ganglia with 4 M GIT could be separated. Thus, HPLC on hydroxyapatite is a rapid and accurate means of quantifying and/or preparing high-molecular-mass RNAs such as t- and ribosomal RNAs.