A total transcriptome profiling method for plasma-derived extracellular vesicles: applications for liquid biopsies

Abstract

Extracellular vesicles (EVs) are key mediators of intercellular communication. Part of their biological effects can be attributed to the transfer of cargos of diverse types of RNAs, which are promising diagnostic and prognostic biomarkers. EVs found in human biofluids are a valuable source for the development of minimally invasive assays. However, the total transcriptional landscape of EVs is still largely unknown. Here we develop a new method for total transcriptome profiling of plasma-derived EVs by next generation sequencing (NGS) from limited quantities of patient-derived clinical samples, which enables the unbiased characterization of the complete RNA cargo, including both small- and long-RNAs, in a single library preparation step. This approach was applied to RNA extracted from EVs isolated by ultracentrifugation from the plasma of five healthy volunteers. Among the most abundant RNAs identified we found small RNAs such as tRNAs, miRNAs and miscellaneous RNAs, which have largely unknown functions. We also identified protein-coding and long noncoding transcripts, as well as circular RNA species that were also experimentally validated. This method enables, for the first time, the full spectrum of transcriptome data to be obtained from minute patient-derived samples, and will therefore potentially allow the identification of cell-to-cell communication mechanisms and biomarkers.

Extracellular RNA profiles with human age

“…A limited number of papers have examined whether circRNAs may be present in serum, mostly using computational methods (Amorim et al., 2017; Gu et al., 2017; Wei et al., 2017). Here, we have used a combination approach and identified 133 extracellular circRNAs (ex‐circRNAs) and further validated age‐related changes in three different ex‐circRNAs: circ_1305 , circ_722, and circ_1445 .…”

“…Our findings are a start at building a bridge over this knowledge gap. In addition, our study validates a recently published approach that incorporates RNA fractionation as a method to capture both short and long RNAs in one sequencing reaction (Amorim et al., 2017), with small differences in the method to fractionate RNA.…”

“…In general, the most well‐studied biotype of exRNA has been microRNAs (miRNAs), but other noncoding RNAs (ncRNAs) are also present in extracellular fluids including Piwi‐interacting RNAs (piRNAs), long noncoding RNAs (lncRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), Y‐RNAs, and circular RNAs (circRNAs) (Amorim et al., 2017; Wei et al., 2017; Yeri et al., 2017). Although not as well‐characterized, fragments or transcripts of messenger RNA (mRNA) have also been identified in extracellular fluid (Amorim et al., 2017; Wei et al., 2017). …”

The Potential Clinical Implications of Circulating Tumor Cells and Circulating Tumor Microemboli in Gastric Cancer

“…Blood is particularly useful in the context of liquid biopsies (LBs), where it allows a series of approaches for the detection of informative markers. In oncology, LBs encompasses three main components: (a) the detection and quantification of cell-free tumor-derived DNA [5][6][7][8]; (b) the quantification and cargo determination of extracellular vesicles [9][10][11]; and (c) the detection, quantification, morphological analysis, and determination of biomarkers in circulating tumor cells (CTCs) [12][13][14][15][16].…”

11th International Symposium on Minimal Residual Cancer (ISMRC): 3–5 May 2018, Montpellier, France

“…Besides CTCs and ctDNA the analysis of circulating microRNAs, exosomes or tumor-educated platelets may provide complementary information as "liquid biopsy" [44]. E.g., the integrin composition of exosomes seems to determine the organ site of metastatic niches [45] and the RNA expression pattern of tumor-educated blood platelets [46] reveals information on tumors in cancer patients.…”