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Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing.

Author(s): Llorens-Revull M, Martínez-González B, Quer J, Esteban JI, Núñez-Moreno G, Mínguez P, Burgui I, Ramos-Ruíz R, Soria ME, Rico A, Riveiro-Barciela M, Sauleda S, Piron M, Corrales I, Borràs FE, Rodríguez-Frías F, Rando A, Ramírez-Serra C, Camós S, Domingo E, Bes M, Perales C, Costafreda MI

Publication: Int J Mol Sci, 2023, Vol. 24, Page

PubMed ID: 37569568 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the size, morphology, and miRNA profile of extracellular vesicles (EVs) isolated from a single plasma specimen using size exclusion chromatography (SEC), iodixanol gradient (GRAD), and a combination of SEC and GRAD. Additionally, miRNA profiles of a single plasma specimen that underwent next-generation sequencing (NGS) library preparation with three different kits were compared. Plasma was separated from the blood of a single volunteer by centrifugation (centrifugation details not provided) and stored at -80°C. Plasma was centrifuged at 1200 g for 20 min at 4°C followed by 10,000 g for 30 min at 4°C before EV isolation.  EVs were isolated from plasma aliquots by: 1) fractionation through qEV2/35 nm columns (SEC), 2) ultracentrifugation at 100,000 g for 3 h at 4°C followed by applying the resuspended pellet to a 40% iodixanol step gradient prepared with OptiPrep and centrifugation at 140,000 g for 18 h at 4°C (GRAD) and 3.) SEC followed by GRAD. Total and subsets of CD9+ EVs were enumerated using a FACSCalibur cytometer.  Total proteins were quantified with the bicinchoninic acid (BCA) assay. Levels of total cholesterol were measured using an AU5800 instrument and levels of apolipoprotein A (apoA) and apolipoprotein B (apoB) were measured using an automated nephelometry system. Particle size and concentration were evaluated by nanoparticle tracking analysis and morphology was visualized by transmission electron microscopy. RNA was extracted from plasma and isolated EVs using the miRNeasy Mini Kit. RNA was quantified using a NanoDrop spectrophotometer and RNA integrity was evaluated using a Bioanalyzer 2100.  RNA was spiked with 52 synthetic miRNAs and sequencing libraries were constructed from plasma RNA using the NEBNext Multiplex Small RNA Library Prep Set for Illumina, the NEXTFlex Small RNA-Seq Kit v3, and the SMARTer smRNA-seq Kit and from EV RNA using the NEBNext Multiplex Small RNA Library Prep Set. Libraries were sequenced using the NextSeq 500/550 High Output Kit v2.5 on a NextSeq 500 system.  

   Summary of Findings:

   Among SEC fractions, levels of CD9 (the EV marker) were highest in fractions 8-12, protein levels were highest in fractions 18-19, total cholesterol was highest in fractions 14-16, and ApoA was highest in fraction 18. VLDL, IDL, LDL, and lipoprotein(a) were not detectable in any of the SEC fractions. Among the GRAD fractions, CD9 was highest in fractions 5-8, protein levels were highest in fractions 3, 4, and 6, and cholesterol ApoA and apoB were undetectable. Fractions from the combined SEC+GRAD method had the lowest protein levels of the EV isolation methods evaluated; for the SEC+GRAD method, CD9 levels were highest in fractions 4-7. When technical replicates were pooled for a given EV isolation method, the fractions with the highest level of CD9 was 8-12 for the SEC method and 4-8 for both the GRAD method, and SEC+GRAD methods. The technical replicate pools for the SEC EV isolation method had more particles/mL than the GRAD or the  SEC+GRAD method (7.49 x10¹¹±5.5x10¹⁰ versus 8.51x10⁹ ±1.98x10⁸ and 3.83 x10⁹±5.53x10⁸, respectively). Both the morphology and the mean particle size of the isolated particles were consistent with EVs (133.37±1.93 nm for SEC pools, 136.5±4 nm for the GRAD pools, and 170.5±4.57 nm for the SEC+GRAD pools) and. Protein levels were comparable between the pools of technical replicates for each EV isolation method and were considerably lower than levels in plasma (1.86 mg/mL for the SEC pool, 2.02 mg/mL for the GRAD pool, and 1.50 mg/mL for the SEC+GRAD pool versus 147 mg/ml in plasma).

   Recovery of the spike-in control miRNA from plasma was most consistent when libraries were constructed with either the NEXT or NEB Kit, but specimens with the spike-in miRNA clustered by library preparation method following principal component analysis. The number of reads in plasma that corresponded to miRNAs was higher in the duplicate NEB (1,653,876 and 1,649,122) and NEXT (4,823,457 and 2,519,523) libraries than the SMARTer libraries (5,348 and 18,918). Significantly more miRNAs were detected in plasma using the NEB or NEXT libraries than the SMARTer libraries (397 or 385, respectively, versus 213, P<0.001). The reproducibility of miRNA detection in plasma between the two technical replicates was higher in NEB libraries than NEXT or SMARTer libraries (84.1% versus 58.7% and 61.5%, respectively).

   Significantly more miRNAs were detected in EVs isolated by the GRAD EV isolation method than SEC or SEC+GRAD (336 versus 223 or 242, respectively, P<0.0001, both) and by SEC+GRAD than SEC (242 versus 223, P=0.037). The highest reproducibility in miRNA detection among the three technical replicates for EV isolation was observed when EVs were isolated using the GRAD method (72.3%) followed by SEC (49.8%) and SEC+GRAD (48.8%) methods.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Flow cytometry
   Cell count/volume	Light scattering
   RNA	Next generation sequencing
   Steroid	Clinical chemistry/auto analyzer
   Lipoprotein	Clinical chemistry/auto analyzer
   Peptide	Colorimetric assay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Next generation sequencing Specific	Technology platform	NEBNext NEXTFlex SMARTer smRNA-seq
   Analyte Extraction and Purification	Analyte isolation method	Size exclusion chromatography (SEC) Iodixanol gradient (GRAD) SEC and GRAD

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