HPLC Analysis of tRNA-Derived Nucleosides

Transfer RNAs (tRNAs), the essential adapter molecules in protein translation, undergo various post-transcriptional modifications. These modifications play critical roles in regulating tRNA folding, stability, and codon-anticodon interactions, depending on the modified position. Methods for detecting modified nucleosides in tRNAs include isotopic labeling combined with chromatography, antibody-based techniques, mass spectrometry, and high-throughput sequencing. Among these, high-performance liquid chromatography (HPLC) has been a cornerstone technique for analyzing modified nucleosides for decades. In this protocol, we provide a detailed, streamlined approach to purify and digest tRNAs from yeast cells and analyze the resulting nucleosides using HPLC. By assessing UV absorbance spectra and retention times, modified nucleosides can be reliably quantified with high accuracy. This method offers a simple, fast, and accessible alternative for studying tRNA modifications, especially when advanced technologies are unavailable. Key features • A streamlined protocol for purifying total tRNAs from yeast cells. • Adaptable for other RNA species and organisms, provided sufficient input material. • Enables the quantification of approximately 20 types of tRNA modifications. • Offers a cost-effective and rapid alternative for analyzing tRNA modifications by HPLC method.