Russo T, Salvatore F, Cimino F
J Chromatogr. 1984 Jul 27;296:387-93. doi: 10.1016/s0021-9673(01)96433-7.
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.
本文描述了一种用于测定水解后的未分级tRNA和酸溶性组织提取物中假尿苷及其他核苷的高效液相色谱法。该方法基于以下步骤：用核糖核酸酶A、蛇毒磷酸二酯酶和细菌碱性磷酸酶的混合物进行tRNA提取和水解；（在酸溶性组织提取物的情况下）通过苯基硼酸凝胶柱上的亲和色谱法进行核苷纯化；通过反相梯度洗脱在十八烷基硅烷柱上进行高效液相色谱法进行核苷分离和定量。该程序可以非常准确地定量假尿苷和其他一些核苷，其灵敏度使得仅需要20微克的tRNA。该方法已被用于比较从正常和淋巴瘤小鼠胸腺中提取的水解tRNA的假尿苷含量，以及相同组织的酸溶性提取物中的假尿苷含量。
