降落PCR技术检测环状RNA表达量的方法评价

Evaluation of the method for detecting circular RNA expression using Touchdown PCR

目的 通过扩增低丰度表达的环状RNA,与常规PCR反应程序比较,评价降落PCR扩增环状RNA的定性定量结果。方法 人肝癌细胞系HepG2细胞中运用Trizol法提取RNA,普通PCR和降落PCR程序扩增circHDAC2片段,琼脂糖凝胶电泳比较两种PCR产物的电泳结果。实时荧光定量PCR(RT-qPCR)对连续5倍稀释的cDNA进行降落PCR程序扩增,比较定量准确度并计算扩增效率。结果 相较于普通PCR,降落PCR程序法能够在不同丰度样本中扩增出circHDAC2,且熔解曲线结果显示单一熔解峰。Sanger测序结果证明扩增产物正确。5倍梯度浓度稀释样本进行降落PCR程序结果显示条带单一,说明降落PCR程序具有良好的敏感性,能够对低丰度circHDAC2进行有效扩增,增效率可达93.27%。结论 降落PCR程序法较普通PCR法扩增circHDAC2基因特异性更高,降落PCR方法在灵敏度、特异度上均优于普通PCR。

Objective By amplifying low-abundance expressed circRNAs,the qualitative and quantitative results of touchdown PCR amplified circRNAs were evaluated compared with conventional PCR reaction procedures.Methods Trizol method was used to extract RNA from human liver cancer cell line HepG2cells.The circHDAC2fragment was amplified by ordinary PCR and touchdown PCR procedures.The electrophoresis results of the two PCR products were compared by agarose gel electrophoresis.Real-time fluorescence quantitative PCR(RT-qPCR)amplifies cDNA serially 5-fold diluted by the landing PCR procedure,compares the quantitative accuracy and calculates the amplification efficiency.Results Compared with ordinary PCR,the touchdown PCR method can amplify circHDAC2in samples with different abundances,and the melting curve results show a single melting peak.Sanger sequencing results proved that the amplified products were correct.The results of the landing PCR procedure on the 5-fold gradient concentration diluted sample showed a single band,indicating that the landing PCR procedure has good sensitivity and can effectively amplify low-abundance circHDAC2,with an amplification efficiency of up to 93.27%.Conclusion The touchdown PCR program method is more specific than the ordinary PCR method for amplifying the circHDAC2gene,and the touchdown PCR method is superior to ordinary PCR in terms of sensitivity and specificity.