目的 建立并优化检测真核细胞mRNA氧化损伤的方法——反转录阻断-双引物扩增法,检测p53 mRNA氧化损伤.方法 建立非细胞体系和细胞体系mRNA氧化损伤模型,提取RNA并反转录成cDNA,根据所检测mRNA的5’端和3’端各设计一对引物,以cDNA为模板进行实时定量PCR.结果 非细胞体系和细胞体系氧化处理组GAPDH和p53 mRNA的氧化损伤程度均明显高于对照组,且随着过氧化氢浓度增加RNA氧化损伤增强；同时一定限度内降低反转录酶用量能提高检测灵敏度.结论 本研究建立并优化的RNA氧化损伤检测方法能检测特定mRNA对氧化的敏感性及损伤程度,为分析相关疾病造成的RNA氧化损伤以及探索其发病机制和治疗方法提供了技术支持.%Objective To develop and optimize the blocking reverse transcription and double-primer method to assay eukaryotic cell mRNA oxidative damage a p53 mRNA oxidative damage. Methods An oxidative mRNA damage model involving both cell-free systems and cell systems was established using hydrogen peroxide. The total RNA was extracted and reversely transcribed into cDNA. According to the detection of mRNA 5' and 3' terminals, two pairs of primers were designed. The real-time quantitative PCR was performed using primers which paired with the 5' terminals or 3'terminals of specific mRNA respectively. Results In both cell-free systems and cell systems of oxidative mRNA damage, the GAPDH and p53 mRNA oxidative damage degree of the group treated by hydrogen peroxide were significantly higher than those in control group, and mRNA oxidative damage was enhanced with the increasing concentration of hydrogen peroxide. At the same time,the detection sensitivity could be improved by reducing the dosage of reverse transcriptase. Conclusion The oxidative mRNA damage detection method developed and optimized in this study can detect specific mRNA oxidative damage, providing technical support for the analysis of pathogenesis of the related diseases.
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