Fetal Bovine Serum RNA Interferes with the Cell Culture derived Extracellular RNA.
Fetal Bovine Serum RNA Interferes with the Cell Culture derived Extracellular RNA.
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DOI：
10.1038/srep31175
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发表时间：
2016-08-09
影响因子：
4.6
通讯作者：
Krichevsky AM
中科院分区：
文献类型：
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作者：
Wei Z;Batagov AO;Carter DR;Krichevsky AM
胎牛血清 (FBS) 几十年来一直用于真核细胞培养。然而，很少有人关注与 FBS RNA 含量对细胞培养物相关的生物学效应。在这里，我们使用 RNA 测序证明 FBS 包含多种蛋白质编码和调节 RNA 种类，包括 mRNA、miRNA、rRNA 和 snoRNA。即使在细胞外囊泡 (EV) 和细胞间通讯研究中常用的囊泡耗尽 FBS (vdFBS) 制剂中进行长时间超速离心后，其中大多数 (>70%) 仍保留。 FBS 相关 RNA 与细胞培养物来源的细胞外 RNA (exRNA) 共分离，并干扰下游 RNA 分析。许多进化上保守的 FBS 衍生 RNA 物种可能被错误地注释为人类或小鼠转录本。值得注意的是，FBS 中丰富的特定 miRNA，如 miR-122、miR-451a 和 miR-1246，之前曾被报道在细胞培养衍生的 EV 中富集，这可能是由于 FBS 的混杂效应所致。对公开的 exRNA 数据集的分析支持了 FBS 污染的概念。此外，FBS 转录物可以被培养的细胞吸收并影响高度敏感的基因表达谱技术的结果。因此，实验设计时必须采取预防措施，以尽量减少 FBS 衍生 RNA 造成的干扰和误解。
Fetal bovine serum (FBS) has been used in eukaryotic cell cultures for decades. However, little attention has been paid to the biological effects associated with RNA content of FBS on cell cultures. Here, using RNA sequencing, we demonstrate that FBS contains a diverse repertoire of protein-coding and regulatory RNA species, including mRNA, miRNA, rRNA, and snoRNA. The majority of them (>70%) are retained even after extended ultracentrifugation in the preparations of vesicle-depleted FBS (vdFBS) commonly utilized in the studies of extracellular vesicles (EV) and intercellular communication. FBS-associated RNA is co-isolated with cell-culture derived extracellular RNA (exRNA) and interferes with the downstream RNA analysis. Many evolutionally conserved FBS-derived RNA species can be falsely annotated as human or mouse transcripts. Notably, specific miRNAs abundant in FBS, such as miR-122, miR-451a and miR-1246, have been previously reported as enriched in cell-culture derived EVs, possibly due to the confounding effect of the FBS. Analysis of publically available exRNA datasets supports the notion of FBS contamination. Furthermore, FBS transcripts can be taken up by cultured cells and affect the results of highly sensitive gene expression profiling technologies. Therefore, precautions for experimental design are warranted to minimize the interference and misinterpretations caused by FBS-derived RNA.