外泌体作为生物标志物携带者，在疾病诊断和预后方面具有巨大的潜力。近年来，外泌体RNA（exoRNA）已成为癌症早期诊断和预后的有前途的候选者，并揭示了在各种疾病中的病理生理学作用。例如，外泌体来源的mRNA、miRNA、circRNA和lncRNA作为信号分子来调节肿瘤生长、血管生成、侵袭转移和对化疗的反应。然而，由于外泌体相对较小的尺寸和外泌体中RNA的量有限，分离高质量和高纯度的外泌体和exoRNA仍然具有挑战性。在本研究中，我们开发了一种新型串联富集方法，基于钛（TiO2）与外泌体和exoRNA脂质双层上的磷酸基之间的特定相互作用，从血清中分离exoRNA。首先在HeLa细胞中证明并优化了基于TiO2的RNA分离方法。在10分钟内，从约5 × 106 HeLa细胞中快速富集出130.9±8.34 μg RNA。相比使用商业的Ultrapure RNA Kit，收率提高了41.5％。随后，使用人类血清进行基于TiO2的exoRNA串联富集，从500 µL人类血清中在30分钟内获得64.53 ± 3.41 ng exoRNA。共鉴定出2137902个reads，包括七种exoRNAs。此方法与各种下游RNA处理技术兼容，不使用苯酚或氯仿等有毒或刺激性试剂，提供了一种简单、经济、快速且安全的从生物样品中提取exoRNA的方法。版权所有©2023 Elsevier公司发表。

Exosomes have great potential as biomarker carriers for disease diagnosis and prognosis. In recent years, exosomal RNA (exoRNA) has become a promising candidate for the early diagnosis and prognosis of cancers, and its pathophysiological roles in various diseases have been revealed. For example, exosome-derived mRNAs, miRNAs, circRNAs, and lncRNAs function as signalling molecules to regulate tumour growth, angiogenesis, invasion, metastasis, and the response to chemotherapy. However, the isolation of exosomes and exoRNA with high quality and purity remains challenging due to the relatively small size of exosomes and the limited amount of RNA in exosomes. In this work, we developed a novel tandem enrichment method to isolate exoRNA from serum based on the specific interaction between titanium dioxide (TiO2) and the phosphate groups on the lipid bilayer of exosomes and of the exoRNA. TiO2-based RNA isolation was first demonstrated and optimized in HeLa cells. A total of 130.9 ± 8.34 µg of RNA was rapidly enriched from approximately 5 × 106 HeLa cells within 10 min. This was a 41.5% higher yield than that using a commercial Ultrapure RNA Kit. TiO2-based tandem enrichment of exoRNA was then performed using human serum, obtaining 64.53±3.41 ng of exoRNA from 500 µL of human serum within 30 min. A total of 2,137,902 reads, including seven types of exoRNAs, were identified from the exosomes. This method is compatible with various downstream RNA processing techniques and does not use toxic or irritating reagents, such as phenol or chloroform, providing a simple, economical, rapid, and safe approach for exoRNA extraction from biological samples.Copyright © 2023. Published by Elsevier B.V.