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通过差异超离心法改善尿小细胞外囊泡的恢复
Ana Teixeira-Marques1, Sara Monteiro-Reis1,2, Diana Montezuma1,3,4
1Cancer Biology and Epigenetics Group, Research Center of IPO Porto (CI-IPOP)/CI-IPOP@RISE (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Center Raquel Seruca (Porto.CCC), R. Dr. António Bernardino de Almeida, 4200-072, Porto, Portugal.
Scientific reports
|May 28, 2024
概括
使用差异超离心法 (UC) 优化尿液细胞外囊 (EV) 隔离可显著改善小EV (SEV) 产量,并保持RNA质量. 这种增强的方法比目前的生物标志物发现协议更节省时间和更有效.
科学领域:
- 生物化学 生物化学
- 细胞生物学 细胞生物学
- 泌尿器科 泌尿器科 泌尿器科 泌尿器科
背景情况:
- 尿液中的细胞外囊泡 (EV) 是包括癌症在内的生殖尿路疾病的宝贵生物标志物.
- 目前用于尿道EV隔离的微分超离心 (UC) 方法是低效的,劳动密集的,并导致显著的EV损失.
- 优化EV分离对于生物标志物发现的临床应用至关重要.
研究的目的:
- 优化差异超离心 (UC) 协议,以加强尿小细胞外囊泡 (SEV) 的隔离.
- 为了减少处理时间,提高SEV产量和RNA完整性.
- 为了将优化的UC协议与现有的方法比较,例如Exoquick-TC和尿液上游过.
主要方法:
- 通过在200,000g下测试各种离心时间 (48分钟,60分钟与25分钟) 来优化差异超离心 (UC),以SEV颗粒化.
- 评估了过和洗尿超的步骤,以评估它们对SEV恢复的影响.
- 优化的UC协议与Exoquick-TC (EXO) 进行了比较,以提高时间效率和SEV收益率.
主要成果:
- 使用48分钟或60分钟离心的优化UC协议显著增加了SEV恢复,而25分钟则相比.
- 过尿超剂增加了1.7倍的SEV回收,而洗步骤降低了0.5倍的产量.
- 优化的UC协议表现出比EXOQUICK-TC更高的时间效率和更大的SEV恢复,同时保持RNA质量和数量.
结论:
- 优化的差异超离心协议提高了尿小细胞外囊泡 (SEV) 隔离的产量和效率.
- 过尿液的超剂是一种有益的步骤,而洗步骤可能会降低SEV产量.
- 这种改进的方法为利用尿路EVs作为生物标志物提供了更节省时间和有效的方法,从而保持关键的RNA完整性.
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