Dataset Information

Serum-free media supplements carry miRNAs that co-purify with extracellular vesicles.

ABSTRACT: 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.

SUBMITTER: Auber M

PROVIDER: S-EPMC6746277 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Serum-free media supplements carry miRNAs that co-purify with extracellular vesicles.

Auber Martin M Fröhlich Dominik D Drechsel Oliver O Karaulanov Emil E Krämer-Albers Eva-Maria EM

Journal of extracellular vesicles 20190909 1

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 se ...[more]

PMID: 31552133

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Project description:From eukaryotes to prokaryotes, all cells secrete extracellular vesicles (EVs) as part of their regular homeostasis, intercellular communication, and cargo disposal. Accumulating evidence suggests that small EVs carry functional small RNAs, potentially serving as extracellular messengers and liquid-biopsy markers. Yet, the complete transcriptomic landscape of EV-associated small RNAs during disease progression is poorly delineated due to critical limitations including the protocols used for sequencing, suboptimal alignment of short reads (20-50 nt), and uncharacterized genome annotations-often denoted as the 'dark matter' of the genome. In this study, we investigate the EV-associated small unannotated RNAs that arise from endogenous genes and are part of the genomic 'dark matter', which may play a key emerging role in regulating gene expression and translational mechanisms. To address this, we created a distinct small RNAseq dataset from human prostate cancer & benign tissues, and EVs derived from blood (pre- & post-prostatectomy), urine, and human prostate carcinoma epithelial cell line. We then developed an unsupervised data-based bioinformatic pipeline that recognizes biologically relevant transcriptional signals irrespective of their genomic annotation. Using this approach, we discovered distinct EV-RNA expression patterns emerging from the un-annotated genomic regions (UGRs) of the transcriptomes associated with tissue-specific phenotypes. We have named these novel EV-associated small RNAs as 'EV-UGRs' or "EV-dark matter". Here, we demonstrate that EV-UGR gene expressions are downregulated by ∼100 fold (FDR < 0.05) in the circulating serum EVs from aggressive prostate cancer subjects. Remarkably, these EV-UGRs expression signatures were regained (upregulated) after radical prostatectomy in the same follow-up patients. Finally, we developed a stem-loop RT-qPCR assay that validated prostate cancer-specific EV-UGRs for selective fluid-based diagnostics. Overall, using an unsupervised data driven approach, we investigate the 'dark matter' of EV-transcriptome and demonstrate that EV-UGRs carry tissue-specific Information that significantly alters pre- and post-prostatectomy in the prostate cancer patients. Although further validation in randomized clinical trials is required, this new class of EV-RNAs hold promise in liquid-biopsy by avoiding highly invasive biopsy procedures in prostate cancer.

Dataset Information

Serum-free media supplements carry miRNAs that co-purify with extracellular vesicles.

Publications

Serum-free media supplements carry miRNAs that co-purify with extracellular vesicles.

Similar Datasets

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