Recent studies on extracellular RNA raised awareness that extracellular vesicles (EVs) isolated from cultured cells may co-purify RNAs derived from media supplements such as fetal bovine serum (FBS) confounding EV-associated RNA. Defined culture media supplemented with a range of nutrient components provide an alternative to FBS addition and allow EV-collection under full medium conditions avoiding starvation and cell stress during the collection period. However, the potential contribution of serum-free media supplements to EV-RNA contamination has remained elusive and has never been assessed. Here, we report that RNA isolated from EVs harvested from cells under serum-replacement conditions includes miRNA contaminants carried into the sample by defined media components. Subjecting unconditioned, EV-free medium to differential centrifugation followed by reverse transcription quantitative PCR (RT-qPCR) on RNA isolated from the pellet resulted in detection of miRNAs that had been classified as EV-enriched by RNA-seq or RT-qPCR of an isolated EV-fraction. Ribonuclease (RNase-A) and detergent treatment removed most but not all of the contaminating miRNAs. Further analysis of the defined media constituents identified Catalase as a main source of miRNAs co-isolating together with EVs. Hence, miRNA contaminants can be carried into EV-samples even under serum-free harvesting conditions using culture media that are expected to be chemically defined. Formulation of miRNA-free media supplements may provide a solution to collect EVs clean from confounding miRNAs, which however still remains a challenging task. Differential analysis of EVs collected under full medium and supplement-deprived conditions appears to provide a strategy to discriminate confounding and EV-associated RNA. In conclusion, we recommend careful re-evaluation and validation of EV small RNA-seq and RT-qPCR datasets by determining potential medium background.

最近有关细胞外RNA的研究提高了人们的意识，即从培养细胞中分离出的细胞外囊泡（EVs）可以共纯化衍生自培养基补充物（例如胎牛血清（FBS））的EV，RNA与EV相关RNA混杂在一起。定义的培养基补充了多种营养成分，是添加FBS的替代方法，并允许在完全培养基条件下进行EV收集，从而避免了收集期间的饥饿和细胞压力。但是，无血清培养基补充剂对EV-RNA污染的潜在贡献仍然难以捉摸，并且从未进行过评估。在这里，我们报道从在血清置换条件下从细胞中收获的电动汽车中分离出的RNA包括通过定义的培养基成分带入样品的miRNA污染物。不受条件限制，不含EV的培养基进行离心分离，然后对从沉淀物中分离的RNA进行逆转录定量PCR（RT-qPCR），从而检测到被RNA-seq或分离的EV-PCR的RT-qPCR归类为EV的miRNA。分数。核糖核酸酶（RNase-A）和去污剂处理去除了大部分但不是全部污染的miRNA。对确定的培养基成分的进一步分析确定了过氧化氢酶是与EV共同分离的miRNA的主要来源。因此，即使在无血清收获条件下，使用预期化学成分确定的培养基，也可以将miRNA污染物带入EV样品中。不含miRNA的培养基补充剂的配制可提供一种解决方案，以从混杂的miRNA中收集纯净的电动汽车，但是这仍然是一项艰巨的任务。在完全培养基和缺乏补充剂的条件下收集的电动汽车的差异分析似乎提供了区分混杂和电动汽车相关RNA的策略。总之，我们建议通过确定潜在的培养基背景，对EV小RNA-seq和RT-qPCR数据集进行仔细的重新评估和验证。