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Comprehensive characterization of human extracellular RNAs (exRNAs) in plasma by high- throughput sequencing

Abstract : Human physiological fluids are known to contain various RNAs outside of the cells either in complexes with proteins (RNPs) or inside of extracellular vesicles. Composition of this extracellular RNA fraction is extremely complex and seems to be modulated in diseases and pathologies. Few studies in the field performed extended analysis of exRNA fraction by microarrays, RT-PCR (qRT-PCR) and deep sequencing, however, no clear consensus on its composition was yet achieved. Indeed, exRNA fraction in human plasma revealed to be highly variable depending on the individual, the sample preparation method or the bioinformatic pipeline used for data analysis. In this study we set up a procedure for plasma preparation, isolation of specific extracellular vesicles, RNA extraction from them, as well as a suitable protocol for library preparation, sequencing and analysis. The goal was to establish a comprehensive list of all RNA species present in plasma, exosome-free plasma and exosomes, taking into account the nature of 5'-and 3'-extremities and thus the biological origin of RNA fragments. The results clearly demonstrate that "native" exRNAs (5'-P and 3'-OH extremities) are mostly represented by miRNAs and Y RNA fragments, while this population becomes a minority when "degradation" fraction (5'-OH, 3'-P or >P) is included into the analysis. "Degraded" RNA fraction mostly contains fragments of rRNAs and other stable RNA species (tRNAs, snRNAs, snoRNAs). In addition to human origin RNAs, exRNA fractions in plasma and in exosomes are enriched in RNAs species coming from symbiotic bacteria, fungi and viruses. These RNAs are mostly prominent in "degraded" RNA fraction and demonstrate an individual-specific composition. Human microbiota is known to play an essential role in many human diseases and our study established experimental protocols for analysis and relationships between human and non-human exRNA fraction in pathologies.
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https://hal.univ-lorraine.fr/hal-02946341
Contributor : Yuri Motorin <>
Submitted on : Monday, September 28, 2020 - 5:40:59 PM
Last modification on : Wednesday, October 14, 2020 - 3:39:58 AM

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Virginie Marchand, Emilie Velot, Yuri Motorin. Comprehensive characterization of human extracellular RNAs (exRNAs) in plasma by high- throughput sequencing. GBM Meeting 2016 CONGRES RNA BIOCHEMIE A BONN, Oct 2016, Bonn, Germany. ⟨hal-02946341⟩

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