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Isolation of extracellular vesicle with different precipitation-based methods exert a tremendous impact on the biomarker analysis for clinical plasma samples.

Author(s): Peng C, Wang J, Bao Q, Wang J., Liu Z, Wen J., Zhang W, Shen Y

Publication: Cancer Biomark, 2020, Vol. , Page

PubMed ID: 32716349 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the yield, size, protein expression, and transcriptome of EVs isolated from pooled plasma using five different precipitation-based kits and ultracentrifugation. Blood was collected from three patients with metastatic osteosarcoma, six patients with non-metastatic primary osteosarcoma, and six healthy patients into acid-citrate-dextrose tubes. Plasma was obtained by centrifugation at 2000 x g for 10 min followed by at 4000 x g for 10 min and was frozen at -80°C. Plasma was pooled by diagnosis. Extracellular vesicles were isolated using ExoQuick Plasma Prep and the Exosome Precipitation Kit, the Total Exosome Isolation Kit, the Wayen Exosome Isolation Kit, the Ribo Exosome Isolation Kit, the miRCURY Exosome Kit, and by ultracentrifugation (centrifugation at 10,000 x g for 20 min, filtration through 0.45 µm filter, centrifugation at 110,000 x g for 11 h, centrifugation at 110,000 x g for 70 min). EVs size and concentration was evaluated using nanoparticle tracking analysis. EV morphology was investigated using transmission electron microscopy (TEM). EVs were characterized by Western Blot analysis using antibodies against CD63, CD81, CD9, TSG101, calnexin, albumin, vimentin, and ApoA1. RNA was extracted using the miRNeasy Micro Kit and analyzed by Tapestation and spectrophotometer. Sequencing libraries were constructed using NEB Next Ultra RNA Library Prep Kit and sequenced using a HiSeq 2500.

   Summary of Findings:

   The appearance of the isolated EV pellets was highly dependent on isolation method with varying degrees of viscosity, opacity, and yellow color observed, but the EVS looked similar under TEM. The size of the EVs isolated ranged from 30-240 nm observed but the median size was not affected by the isolation method. The yield of EVs was highly dependent on isolation method (Friedman test P=0.0236) with the highest levels obtained using the Wayen Exosome Isolation Kit or Ribo Exosome Isolation Reagent and the lowest using ultracentrifugation. The co-precipitated protein levels also differed among the isolation methods (P=0.0165) with the highest levels when EVs were isolated using miRCURY Exosome Kits or Ribo Exosome Isolation Reagent followed by the other precipitation-based techniques and lowest using ultracentrifugation. While the surface markers CD63 and CD8 were detected in all isolates, calnexin was not detected, indicating the absence of cellular contamination, regardless of isolation method. Vimentin, a marker of sarcoma, was detected in the specimens from patients regardless of isolation method but not the control. In contrast, levels of CD9 and TSG-101 were significantly affected by isolation method (P=0.0124 and P=0.0305, respectively) and indicated differences in EV subpopulation among the methods. Further, levels of the plasma proteins albumin and ApoA1 differed significantly among methods (P=0.0179 and P=0.0262, respectively). The yield and integrity of evRNA was not affected by isolation method, but EVRNA integrity was modestly correlated with maximum EV size (R2=0.54, P=0.0389). The 5’ to 3’ UTR coverage and the UTR to coding ratios were dependent on the isolation method used. Further, the percentage of short non-coding RNA, pseudogenes, long noncoding RNA, and protein-coding RNA were dependent on the isolation method with extraction by ultracentrifugation or Wayen Exosome Isolation Kit followed by miRCURY Exosome Kits resulting in more short non-coding genes and pseudogenes and less long non-coding and protein-coding genes. Hierarchical clustering clustered the specimens somewhat by isolation method rather than patient source with all specimens isolated using the Qiagen Kit clustering together.  Importantly, the variability in gene expression among the kits was greater than the variability between the specimens. When the list of 50 most abundant RNAs were compared among kits, only nine RNAs were found on each of the lists in plasma from metastatic osteosarcoma patients and 14 were found on each list in plasma from healthy patients. Gene expression variability among the three specimens was higher when extraction was by ultracentrifugation then the precipitation-based methods, which the authors state indicates that the precipitation-based methods failed to capture the true biological variability.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal
   • Neoplastic - Sarcoma

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Light scattering
   Morphology	Electron microscopy
   Morphology	Macroscopic observation
   RNA	Automated electrophoresis/Bioanalyzer
   Lipoprotein	Western blot
   RNA	Next generation sequencing
   RNA	Spectrophotometry
   Protein	Western blot

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Healthy Non-metastatic primary osteosarcoma Metastatic osteosarcoma
   Analyte Extraction and Purification	Analyte isolation method	ExoQuick Plasma Prep and the Exosome Precipitation Kit Total Exosome Isolation kit Wayen Exosome Isolation kit Ribo Exosome Isolation kit miRCURY Exosome Kit Ultracentrifugation

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