Lan Lin, Guo Qiuping, Nie Hemin, Zhou Chang, Cai Qingyun, Huang Jin, Meng Xiangxian
College of Biology , Hunan University , Changsha , P. R. China . Email:
State Key Laboratory of Chemo/Biosensing and Chemometrics , P. R. China.
Chem Sci. 2018 Dec 4;10(7):2034-2043. doi: 10.1039/c8sc04621b. eCollection 2019 Feb 21.
Here, we present a highly specific, sensitive and cost-effective system to quantify microRNA (miRNA) expression based on two-step RT-qPCR with EvaGreen detection chemistry, called linear-hairpin variable primer RT-qPCR. It takes advantage of the novel designed variable primer, which is initially designed to be linear, extending to form a hairpin structure and replacing the target miRNA for cyclic RT. Then the RT product is quantified by conventional EvaGreen based qPCR. The results show that this method has a dynamic range of 8 logs and the sensitivity is sufficient to directly detect down to 4 target miRNA molecules with a total analysis time of less than 2 hours. It is capable of discriminating between similar miRNAs, leading to an accurate representation of the mature miRNA content in a sample. The RT step can be multiplexed and the 8 miRNA profiles measured in 7 mouse tissues by this method show an excellent correlation with the commercial standard TaqMan RT-qPCR assays ( = 0.9881).
在此，我们展示了一种基于两步逆转录定量聚合酶链反应（RT-qPCR）与EvaGreen检测化学方法的高度特异性、灵敏且经济高效的系统，用于定量微小RNA（miRNA）表达，称为线性发夹可变引物RT-qPCR。它利用了新设计的可变引物，该引物最初设计为线性，延伸形成发夹结构并替代目标miRNA进行循环逆转录。然后通过基于传统EvaGreen的定量聚合酶链反应对逆转录产物进行定量。结果表明，该方法的动态范围为8个数量级，灵敏度足以直接检测低至4个目标miRNA分子，总分析时间不到2小时。它能够区分相似的miRNA，从而准确呈现样本中成熟miRNA的含量。逆转录步骤可以进行多重分析，通过该方法在7种小鼠组织中测量的8种miRNA谱与商业标准TaqMan RT-qPCR检测结果显示出极好的相关性（r = 0.9881）。
