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Abstract
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.
Highlights
Deep sequence analysis of extracellular RNA released by cultured cells in forms of extracellular vesicles (EVs) and lipoprotein complexes (RNPs) is an expanding area of research
Another specific microRNA, miR-451a, expressed in diverse cells and tissues with highest abundance in the blood[2], was reported as the most enriched miRNA in EVs secreted by various cell types[5,6,7,8,9,10,11]
Consistent with these findings, we found miR-451a enriched in the pelleted EVs isolated from the conditioned media of U251 and 20/3 glioma monolayer cultures grown in DMEM/10% vesicle-depleted FBS (vdFBS) (Fig. 1a)
Summary
Extracellular RNA received: 04 April 2016 accepted: 13 July 2016 Published: 09 August 2016. FBS-associated RNA is co-isolated with cell-culture derived extracellular RNA (exRNA) and interferes with the downstream RNA analysis. ExRNA may reflect the entire cellular transcriptome, a specific RNA class (e.g. miRNA), or one of the steps of RNA metabolism, and provide a peripheral measure for monitoring the cellular transcriptome. Addressing this question is critical for the study of intercellular communication and development of RNA biomarkers. Many reports characterized exRNA isolated from human and mouse serum and suggested serum RNA as the major source of disease biomarkers[1], little attention has been paid to confounding effects of FBS-derived RNA on cell-culture based studies. We characterize the RNA content of FBS and vesicle-depleted FBS (vdFBS), and demonstrate its interference with cell-derived exRNA isolated from conditioned media and, possibly, cellular RNA
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