Xu Shengqiang, Nasr Seyedmehdi Hossaini, Chen Daoyang, Zhang Xiaoxian, Sun Liangliang, Huang Xuefei, Qian Chunqi
Department of Radiology, 846 Service Rd, East Lansing, MI, 48824.
Department of Chemistry, 578 S Shaw Ln, East Lansing, MI, 48824.
ACS Biomater Sci Eng. 2018 Feb 12;4(2):654-662. doi: 10.1021/acsbiomaterials.7b00668. Epub 2017 Dec 18.
MicroRNA (miRNA) in urine has been considered as a potential biomarker for early-stage diagnosis of multiple diseases like urinary system cancer, kidney injury and diabetes, owing to their many demonstrated advantages including long-term stability and noninvasiveness. However, the traditional enrichment and extraction processes of miRNAs from urine are cumbersome and tedious due to the low concentration and multiple carriers of miRNAs. Herein, we present a novel method to collect low concentrations of miRNAs from dilute solutions such as urine and cell culture medium. 10-nm core sized magnetic nanoparticles with carboxylic acid coating can adsorb low-concentration proteins, and form protein corona which makes them easy to aggregate and precipitate for subsequent isolation. In urine and cell culture medium, these nanoparticles can aggregate with proteins, including miRNAs-associated protein Argonaute 2 and microvesicle-related proteins, to form precipitates, so that miRNAs can be easily extracted from pellets by small amount of lysis buffer for subsequent analysis such as real-time PCR. Our method provides a facile way to enrich miRNAs from biofluids without the need of ultracentrifugation and immunoprecipitations, bringing remarkable convenience to miRNAs-based biomarker research.
尿液中的微小RNA（miRNA）因其诸多已被证实的优势，如长期稳定性和非侵入性，而被视为多种疾病（如泌尿系统癌症、肾损伤和糖尿病）早期诊断的潜在生物标志物。然而，由于miRNA在尿液中的浓度较低且存在多种载体，传统的从尿液中富集和提取miRNA的过程繁琐且乏味。在此，我们提出了一种从稀释溶液（如尿液和细胞培养基）中收集低浓度miRNA的新方法。具有羧酸涂层的10纳米核心尺寸的磁性纳米颗粒可以吸附低浓度蛋白质，并形成蛋白质冠层，这使得它们易于聚集和沉淀以便后续分离。在尿液和细胞培养基中，这些纳米颗粒可以与蛋白质（包括与miRNA相关的蛋白质AGO2和微囊泡相关蛋白质）聚集形成沉淀，从而可以通过少量裂解缓冲液从沉淀中轻松提取miRNA，用于后续分析，如实时PCR。我们的方法提供了一种无需超速离心和免疫沉淀即可从生物流体中富集miRNA的简便方法，为基于miRNA的生物标志物研究带来了极大的便利。
