摘要
目的以微小RNA(miRNA)作为检测样本,考察影响实时荧光定量聚合酶链反应(RT-qPCR)检测结果的关键点,并筛选出最优的检测方法。方法采用3种miRNA提取方法(分别使用EasyPure^(■)miRNA提取试剂盒、miRNA提取试剂盒、TransZol Up Plus RNA提取试剂盒)和3种反转录方法(分别使用TransScript^(■)第一链cDNA反转录试剂盒、Evo M-MLV反转录试剂盒、miRNA第一链cDNA合成试剂盒)处理大鼠纹状体脑区,检测miRNA与c DNA的质量和效率,并使用常规RT-qPCR检测miRNA水平,比较不同方法所得结果。结果3种RNA提取方法得到的miRNA质量和效率比较差异均有统计学意义,其中方法2的效果较好,但方法1、2、3的RT-qPCR结果比较差异无统计学意义(miR-132:25.91±9.79、25.26±10.25、27.28±7.39,miR-U6:27.98±11.25、25.98±9.78、29.62±9.65,均P>0.05);3种反转录方法所得实验结果比较差异有统计学意义,方法3所得结果明显低于方法1、方法2(miR-132:16.53±3.17比35.20±1.06、31.42±2.95,miR-U6:16.63±1.73比36.06±2.57、35.59±1.54,均P<0.05),主要影响RT-qPCR的扩增效率和扩增特异性。结论使用能高效富集约18 nt大小RNA的提取方法和在miRNA的3’末端加多聚A尾(Poly A)的反转录酶,可以得到更可靠的RT-qPCR结果。
Objective To investigate the key points that affect the results of real-time fluorescence quantitative polymerase chain reaction(RT-qPCR),and to select the optimal detection method using micro RNA(miRNA)as the detection sample.Methods Three miRNA extraction methods(including EasyPure^(■)miRNA Kit,miRNeasy Mini Kit,TransZol Up Plus RNA Kit)were used and three reverse transcription methods(including TransScript^(■)All-in-One First-Strand cDNA Synthesis SuperMix for qPCR,Evo M-MLV reverse transcription kit,and All-in-One miRNA First-Strand cDNA)were used to treat the brain region of rat striatum,detect the quality and efficiency of miRNA and cDNA.The content of miRNA by conventional RT-qPCR method was detected,and the results obtained by different methods were compared.Results There were significant differences in the quality and efficiency of miRNA obtained by three RNA extraction methods.Among them,method 2 had a better effect,but there was no significant difference in the RT-qPCR results of methods 1,2 and 3(miR-132:25.91±9.79,25.26±10.25,27.28±7.39,miR-U6:27.98±11.25,25.98±9.78,29.62±9.65,all P>0.05).The experimental results obtained by the three reverse transcription methods showed statistically significant differences,with the results obtained by method 3 being significantly lower than those obtained by methods 1 and 2(miR-132:16.53±3.17 vs.35.20±1.06,31.42±2.95,miR-U6:16.63±1.73 vs.36.06±2.57,35.59±1.54,all P<0.05),which mainly affected the amplification efficiency and specificity of RT-qPCR.Conclusion Using an extraction method that could efficiently enrich approximately 18 nt sized RNA and adding poly A tail reverse transcriptase at the 3'end of miRNA could obtain more reliable RT-qPCR results.
作者
付瑶
刘海婷
施俊超
栾天
文静
张俊
张大军
Fu Yao;Liu Haiting;Shi Junchao;Luan Tian;Wen Jing;Zhang Jun;Zhang Dajun(School of Pharmacy,Shenyang Medical College,Shenyang 110000,Liaoning,China)
出处
《实用检验医师杂志》
2023年第2期193-197,共5页
Chinese Journal of Clinical Pathologist
基金
辽宁省科技厅自然科学基金项目(2020-MS-313)
沈阳医学院大创项目(20198030)。
关键词
实时荧光定量聚合酶链反应
微小RNA
提取方法
反转录方法
Real time quantitative polymerase chain reaction
Micro RNA
Extraction method
Reverse transcription method
