血浆miRNA快速制备方法建立和优化

Establishment and optimization of a method to extract miRNAs from plasma

目的建立并优化用于血浆microRNA(miRNA)检测分析的miRNA快速提取方法。方法使用含表面活性剂的提取液和加热方法提取血浆miRNA,采用RNA酶抑制剂抑制RNA降解,利用检测miRNA的poly(A)加尾、逆转录反应和实时定量PCR法检测血浆miRNA表达水平,评估方法的可行性。结果成功建立了提取液和加热提取miRNA一步法,实时定量PCR结果表明该方法具有良好的扩增特异性、可重复性,方法稳定可靠。结论提取液和加热一步法简便、快速、低成本,血浆用量少,为临床血浆miRNA检测分析提供了一种新的技术方法。

Objective To develop and optimize a new method to extract miRNAs from plasma. Methods miRNAs were extracted from plasma by mixing it with the extraction solution that contained surfactant and by heating. Then the ribonuclease inhibitor was added into the extraction to prevent RNAs from degradation. The expression level of each miRNA was detected by real-time quantitative PCR in oder to evaluate the feasibility of this method. Results A method which extracted miRNAs from plamsa in just one step was established. The specificity,reproducibility and stability of this method have been demonstrated by real-time quantitative PCR. Conclusion The one-step method is simple,inexpensive,and plasma-saving. It seems like a new method for clinical examination of miRNAs from plasma.