An improved method for detecting circulating microRNAs with S-Poly(T) Plus real-time PCR.
An improved method for detecting circulating microRNAs with S-Poly(T) Plus real-time PCR.
复制标题
使用 S-Poly(T) Plus 实时 PCR 检测循环 microRNA 的改进方法
DOI：
10.1038/srep15100
复制
发表时间：
2015-10-13
影响因子：
4.6
通讯作者：
Gou D
中科院分区：
文献类型：
--
作者：
Niu Y;Zhang L;Qiu H;Wu Y;Wang Z;Zai Y;Liu L;Qu J;Kang K;Gou D
本文介绍了一种简单、灵敏、特异的循环微RNA分析方法，称为S-Poly(T)Plus荧光定量聚合酶链式反应方法。这种新方法是在我们之前开发的S-Poly(T)方法的基础上建立的，在逆转录过程中使用了独特的S-Poly(T)引物，以提高灵敏度和特异性。与S-Poly(T)方法相比，S-Poly(T)Plus通过一步、多阶段反应进一步提高了灵敏度和简单性，其中RNA被多腺化并同时反转录。改进的S/P miRsol血清/血浆总RNA提取方法进一步提高了循环miRNA检测的灵敏度，该方法利用糖原提高RNA产量。我们通过定量检测与先天性心脏病相关的肺动脉高压患者血清中miRNA的表达谱来验证我们的方法。综上所述，我们建立了一种简单、灵敏和特异的检测循环 μ的方法，可以检测100份血清或血浆中的266个miRNA。该方法为基于miRNA生物标志物的基础miRNA研究和人类疾病的临床诊断提供了一种很有前途的工具。
We herein describe a simple, sensitive and specific method for analysis of circulating microRNAs (miRNA), termed S-Poly(T) Plus real-time PCR assay. This new method is based on our previously developed S-Poly(T) method, in which a unique S-Poly(T) primer is used during reverse-transcription to increase sensitivity and specificity. Further increased sensitivity and simplicity of S-Poly(T) Plus, in comparison with the S-Poly(T) method, were achieved by a single-step, multiple-stage reaction, where RNAs were polyadenylated and reverse-transcribed at the same time. The sensitivity of circulating miRNA detection was further improved by a modified method of total RNA isolation from serum/plasma, S/P miRsol, in which glycogen was used to increase the RNA yield. We validated our methods by quantifying miRNA expression profiles in the sera of the patients with pulmonary arterial hypertension associated with congenital heart disease. In conclusion, we developed a simple, sensitive, and specific method for detecting circulating miRNAs that allows the measurement of 266 miRNAs from 100 μl of serum or plasma. This method presents a promising tool for basic miRNA research and clinical diagnosis of human diseases based on miRNA biomarkers.