胎牛血清RNA干擾了細(xì)胞培養(yǎng)外源性RNA

幾年前提出，并在該領(lǐng)域仍然高度爭(zhēng)議的一個(gè)重要問(wèn)題是，是否特異性RNA種類或圖案在電動(dòng)汽車和的RNP充實(shí)。這種富集將支持RNA分泌的積極和監(jiān)管機(jī)制。可替代地，exRNA可以反映整個(gè)蜂窩轉(zhuǎn)錄，特定的RNA類（如miRNA）的，或RNA的代謝的步驟之一，因此，對(duì)于監(jiān)視蜂窩轉(zhuǎn)錄提供一個(gè)外圍設(shè)備的措施。解決這個(gè)問(wèn)題是細(xì)胞間通訊和RNA的生物標(biāo)志物的發(fā)展研究是至關(guān)重要的。細(xì)胞培養(yǎng)系統(tǒng)exRNA生物合成的研究使用和釋放，在很大程度上依賴于胎牛血清（FBS），常用的試劑眾多類型細(xì)胞的生長(zhǎng)至關(guān)重要。盡管許多報(bào)道特點(diǎn)exRNA從人類和小鼠血清中分離和血清建議RNA作為疾病生物標(biāo)志的主要來(lái)源1，很少有人注意支付給FBS衍生的RNA的基于細(xì)胞培養(yǎng)研究混雜效應(yīng)。在這里，我們描述的FBS的RNA含量以及囊泡-貧化的FBS（vdFBS），并證明其與細(xì)胞衍生exRNA從條件培養(yǎng)基，可能的話，細(xì)胞RNA分離的干擾。

英文原文：

Fetal  Bovine Serum RNA Interferes with the Cell Culture derived Extracellular RNA

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.

Introduction

Deep sequence analysis of extracellular RNA (exRNA) released by cultured cells in forms of extracellular vesicles (EVs) and lipoprotein complexes (RNPs) is an expanding area of research. An important question raised years ago and still highly debated in the field, is whether specific RNA species or motifs are enriched in EVs and RNPs.