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Assessment of small RNA sorting into different extracellular fractions revealed by high-throughput sequencing of breast cell lines.

ABSTRACT: Intercellular communication can be mediated by extracellular small regulatory RNAs (sRNAs). Circulating sRNAs are being intensively studied for their promising use as minimally invasive disease biomarkers. To date, most attention is centered on exosomes and microRNAs as the vectors and the secreted species, respectively. However, this field would benefit from an increased understanding of the plethora of sRNAs secreted by different cell types in different extracellular fractions. It is still not clear if specific sRNAs are selected for secretion, or if sRNA secretion is mostly passive. We sequenced the intracellular sRNA content (19-60 nt) of breast epithelial cell lines (MCF-7 and MCF-10A) and compared it with extracellular fractions enriched in microvesicles, exosomes and ribonucleoprotein complexes. Our results are consistent with a non-selective secretion model for most microRNAs, although a few showed secretion patterns consistent with preferential secretion. On the contrary, 5' tRNA halves and 5' RNA Y4-derived fragments of 31-33 were greatly and significantly enriched in the extracellular space (even in non-mammary cell lines), where tRNA halves were detected as part of ?45 kDa ribonucleoprotein complexes. Overall, we show that different sRNA families have characteristic secretion patterns and open the question of the role of these sRNAs in the extracellular space.

SUBMITTER: Tosar JP

PROVIDER: S-EPMC4477662 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Project description:Extracellular vesicles (EVs) have great potential as a source for clinically relevant biomarkers since they can be readily isolated from biofluids and carry microRNA (miRNA), mRNA, and proteins that can reflect disease status. However, the biological and technical variability of EV content is unknown making comparisons between healthy subjects and patients difficult to interpret. In this study, we sought to establish a laboratory and bioinformatics analysis pipeline to analyse the small RNA content within EVs from patient serum that could serve as biomarkers and to assess the biological and technical variability of EV RNA content in healthy individuals. We sequenced EV small RNA from multiple individuals (biological replicates) and sequenced multiple replicates per individual (technical replicates) using the Illumina Truseq protocol. We observed that the replicates of samples clustered by subject indicating that the biological variability (~95%) was greater than the technical variability (~0.50%). We observed that ~30% of the sequencing reads were miRNAs. We evaluated the technical parameters of sequencing by spiking the EV RNA preparation with a mix of synthetic small RNA and demonstrated a disconnect between input concentration of the spike-in RNA and sequencing read frequencies indicating that bias was introduced during library preparation. To determine whether there are differences between library preparation platforms, we compared the Truseq with the Nextflex protocol that had been designed to reduce bias in library preparation. While both methods were technically robust, the Nextflex protocol reduced the bias and exhibited a linear range across input concentrations of the synthetic spike-ins. Altogether, our results indicate that technical variability is much smaller than biological variability supporting the use of EV small RNAs as potential biomarkers. Our findings also indicate that the choice of library preparation method leads to artificial differences in the datasets generated invalidating the comparability of sequencing data across library preparation platforms.

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Assessment of small RNA sorting into different extracellular fractions revealed by high-throughput sequencing of breast cell lines.

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