Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell‐free blood

Abstract

IntroductionmicroRNA (miRNA) are small non-coding RNA species that are transcriptionally processed in the host cell and released extracellularly into the bloodstream. Normally involved in post-transcriptional gene silencing, the deregulation of miRNA has been shown to influence pathogenesis of a number of diseases.BackgroundNext-generation deep sequencing (NGS) has provided the ability to profile miRNA in biological fluids making this approach a viable screening tool to detect miRNA biomarkers. However, collection and handling procedures of blood needs to be greatly improved for miRNA analysis in order to reliably detect differences between healthy and disease patients. Furthermore, ribonucleases present in blood can degrade RNA upon collection rendering extracellular miRNA at risk of degradation. These factors have consequently decreased sensitivity and specificity of miRNA biomarker assays.MethodsHere, we use NGS to profile miRNA in various blood components and identify differences in profiles within peripheral blood compared to cell-free plasma or serum and extracellular vesicles known as exosomes. We also analyse and compare the miRNA content in exosomes prepared by ultracentrifugation methods and commercial exosome isolation kits including treating samples with RNaseA.ConclusionThis study demonstrates that exosomal RNA is protected by RNaseA treatment and that exosomes provide a consistent source of miRNA for disease biomarker detection.

Evaluation of serum extracellular vesicle isolation methods for profiling miRNAs by next‐generation sequencing

“…Should this be the case, additional validation of sepsis-related miRNA signatures might be carried out on total cell-free RNA without prior enrichment of EVs, reducing time and cost of analysis. Even though exosomes have been shown to provide an enriched source of miRNA with higher predictive value than total cell-free blood, miRNAs of diagnostic potential might be associated with different carriers in a disease-specific manner, calling for the careful validation of previous findings in each biomarker discovery process [2,94]. In diseases with less drastic clinical manifestation than sepsis, extracellular signalling could be more clearly detectable in pure EVs as opposed to crude preparations of cell-free RNA.…”

“…Several previous publications reported the feasibility and utility of sequencing small RNA in EVs isolated from serum, plasma, urine, and cell culture supernatant [2,27–30]. Small RNA-Seq experiments often focus on valuable applications such as liquid biopsy-based diagnostics and, consequently, clinical samples.…”

Gestational Diabetes Mellitus Is Associated With Changes in the Concentration and Bioactivity of Placenta-Derived Exosomes in Maternal Circulation Across Gestation

“…Indeed, it has become clear that "cell-free miRNA and RNA" in plasma (that are currently used in prenatal diagnosis) are contained within the exosome. Recently, it has been established that small RNA (including miRNAs) contained within exosomes are protected against RNase treatment (32). Interestingly, unique and common miRNA between plasma and PdEs was reported.…”

Summary of the ISEV workshop on extracellular vesicles as disease biomarkers, held in Birmingham, UK, during December 2017

“…Lesley Cheng (La Trobe University, Australia) emphasised the relevance of pre-analytical standardisation of blood and urine sample collection and handling on EVs, in particular for isolation of EV-associated micro (mi)RNA [20]. Importantly, even when the effect of pre-analytical variables on the sample are unknown, consistency and compliance with downstream SOPs is extremely important to enable the comparison of results between collected samples.…”