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Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS
Published on: February 24, 2021
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HPLC Analysis of tRNA-Derived Nucleosides
Xingxing Chen1,2, Fu Xu3
1Department of Radiation Oncology, Fudan University Shanghai Cancer Center, Shanghai, China.
Bio-Protocol
|March 3, 2025
Summary
This study presents a streamlined method for analyzing transfer RNA (tRNA) modifications in yeast. The protocol uses high-performance liquid chromatography (HPLC) for accurate quantification of modified nucleosides, offering a cost-effective alternative.
Area of Science:
- Molecular Biology
- Biochemistry
Background:
- Transfer RNAs (tRNAs) are crucial for protein translation.
- Post-transcriptional modifications regulate tRNA structure and function.
- Accurate detection of modified nucleosides is vital for understanding tRNA biology.
Purpose of the Study:
- To provide a detailed, streamlined protocol for tRNA purification and nucleoside analysis.
- To enable accurate quantification of modified nucleosides using HPLC.
- To offer an accessible method for studying tRNA modifications.
Main Methods:
- Purification of total tRNAs from yeast cells.
- Digestion of purified tRNAs into nucleosides.
- Analysis of nucleosides using high-performance liquid chromatography (HPLC).
- Quantification based on UV absorbance spectra and retention times.
Main Results:
- A streamlined protocol for tRNA purification from yeast.
- Reliable quantification of approximately 20 types of modified nucleosides.
- Demonstrated accuracy and accessibility of the HPLC method.
Conclusions:
- The developed HPLC method is a simple, fast, and cost-effective approach for analyzing tRNA modifications.
- This protocol is adaptable for other RNA species and organisms.
- It serves as a valuable tool, especially when advanced technologies are limited.
