Determination of pseudouridine in tRNA and in acid-soluble tissue extracts by high-performance liquid chromatography

A high-performance liquid chromatographic method to measure pseudouridine and other nucleosides in hydrolyzed unfractionated tRNA and in acid soluble tissue extracts is described. The method is based on the following steps: tRNA extraction and hydrolysis by a mixture of ribonuclease A, snake venom phosphodiesterase and bacterial alkaline phosphatase; nucleoside purification (in the case of acid soluble tissue extract) by affinity chromatography on a phenyl-boronate gel column; nucleoside separation and quantitation by high-performance liquid chromatography on an octadecylsilane column by a reversed polarity gradient elution. The procedure allows a very accurate quantitation of pseudouridine and some other nucleosides, and its sensitivity is such that only 20 micrograms of tRNA are required. The method has been utilized to compare the pseudouridine content of hydrolyzed tRNA extracted from normal and lymphomatous murine thymus, as well as the pseudouridine content in acid soluble extracts from the same tissues.