走向标准化表体转录组分析:在人类RNA中检测和量化改性核糖核酸的质谱测量和量化的实验室间比较
Martin Hengesbach1, Chi-Kong Chan2, Tulsi Bhandari3
1Institute for Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University, Mainz, 55128, Germany.
Nucleic acids research
|September 18, 2025
概括
这项研究协调了跨实验室的RNA修饰分析. 多个实验室对液态染色学结合的并联四极质谱 (LC-MS/MS) 工作流程进行了比较,建立了一个可重复的表谱组分析平台.
科学领域:
- 分子生物学分子生物学
- 生物化学 生物化学
- 基因组学就是基因组学.
背景情况:
- 人类RNome包括RNA及其化学修饰 (表转录组).
- 了解RNA修饰对于基因表达和细胞表型调节至关重要.
- 使用液态染色学结合的双重四极质谱法 (LC-MS/MS) 精确量化改性核酸是必不可少的,但由于可重现性问题而受到阻碍.
研究的目的:
- 在多个实验室中比较和协调基于LC-MS/MS的RNA修饰分析的工作流程.
- 在表皮转录组分析中识别影响复制性的因素.
- 建立一个强大的和可转移的RNA修饰量化分析平台.
主要方法:
- 一个涉及三个实验室分析相同的总RNA样本的多实验室比较.
- 标准化样品运输,RNA水解,LC-MS/MS分析和数据处理的协议.
- 使用LC-MS/MS检测和定量修饰的核糖核酸.
主要成果:
- 在不同的协议和运营商中,连续检测和量化了17种RNA修饰.
- 七种修改显示对实验条件,污染或核糖核酸不稳定性的敏感性,影响精度.
- 实验室之间发现了强烈的一致性,相对量化变化系数为20%,绝对量化变化系数为10%.
结论:
- 建立了一个强大的和可采用的表表体转录组分析平台.
- 开发的平台使得可靠和可重复的RNA修饰量化在实验室.
- 协调的工作流对于推进RNA修饰及其功能的研究至关重要.
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