优化和验证人精浆miRNAs提纯方法

Optimization and verification of purification methods of miRNAs in human seminal plasma

目的优化精浆miRNAs提纯方法,为后续实验奠定基础。方法收集国家卫生计生委科研所生殖健康服务中心正常精液样本5例,非梗阻性无精子症(NOA)患者(经北京大学第三医院检查确诊)精液样本22例。根据不同RNA提取方法分为4组:Trizol LS组、蛋白酶K+Trizol LS组、miRNeasy Micro kit组及Trizol LS+miRNeasy Micro kit联合使用组(改良组)。比较4组提纯NOA患者精浆RNA的OD260/280,使用Agilent2100生物分析仪检测改良法提纯精浆miRNAs的纯度和质量,采用实时定量PCR方法比较4组精浆miR-106b的溶解曲线,并比较精液参数正常组(n=5)和NOA患者组(n=10)精液样本中的miR-106b的表达量。结果改良组提纯精浆RNA的OD260/280为(1.90±0.03)显著高于其他3组(P<0.05);改良组提纯精浆miRNAs的完整数合格,色谱图位置特异性明显,峰值清晰;4组miR-106b的溶解曲线中,改良组曲线符合标准;NOA患者精浆中miR-106b的表达丰度显著低于参数正常组(P<0.01)。结论Trizol LS与miRNeasy Micro kit联合使用提纯的精浆miRNAs质量好,能满足后续实时定量PCR、深度测序等实验要求。小样本分析发现NOA患者精浆miR-106b含量显著低于精液参数正常组,有待大样本验证其是否能作为临床诊断NOA的分子指标。

Objective： To optimize the method for purification of miRNAs in seminal plasma to provide the foundation for further experiments. Methods： According to different RNA extraction methods, the treated semen samples were divided into 4 groups： Trizol LS group （control group） used Trizol LS method; Protease K＋Trizol LS group was firstly treated with Protease K, and then used Trizol LS method to extract RNA; QIAGEN miRNeasy Micro kit group used QIAGEN miRNeasy Micro kit; Trizol LS ＋ QIAGEN miRNeasy Micro kit combination group （modified group） was firstly treated with Trizol LS reagent,and then used QIAGEN miRNeasy Micro kit. The purity and quality of the extracted miRNAs were determined by NANO DROP, real-time quantitative PCR was performed to detect the level of miR-106b in normal group （n=5） and non- obstructive azoospermia （n= 10）. Results： The miRNA OD260/280 of the modified group [（1.90 ±0.03）] was significantly higher than that of the other three groups （P〈0. 05）. Real-time quantitative PCR detection of miR-106b showed that the peaks of the first three groups were not single, non-specific amplification, but the fourth group had a normal dissolution curve. The purified miRNAs of the fourth group was detected by the Aglient 2100 bioanalytical system,which was qualified to meet the requirements for deep sequencing. The content of miR-106b in seminal plasma of patients with non-obstructive azoospermia was significantly lower than that in normal group （P〈0. 01）. Conclusion： Trizol LS combined with miRNeasy Micro kit for purifying seminal plasma miRNA is the best way to meet the follow-up real-time quantitative PCR, depth sequencing and other experimental requirements. Analysis of small samples showed that the content of miR-106b in seminal plasma of patients with non-obstructive azoospermia was significantly lower than that in semen parameters.