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Enrichment of Exosome-Like Extracellular Vesicles from Plasma Suitable for Clinical Vesicular miRNA Biomarker Research.

Author(s): Moon S, Shin DW, Kim S, Lee YS, Mankhong S, Yang SW, Lee PH, Park DH, Kwak HB, Lee JS, Kang JH

Publication: J Clin Med, 2019, Vol. 8, Page

PubMed ID: 31731761 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to investigate the effects of plasma volume, proteinase K treatment, acidification, and extraction method on the total protein yield, abundance of ELV and non-ELV proteins, and ELV morphology and size distribution. The abundance of three miRNAs were compared between plasma and ELV specimens. Fasting blood was drawn by venipuncture from an unspecified number of healthy volunteers and 6 elderly patients (3 with mild Alzheimer’s disease and 3 cognitively normal) into EDTA tubes. Plasma was obtained by centrifugation at 3000 x g for 10 min at room temperature followed by removal of platelets and cellular debris through centrifugation at 10,000 x g for 20 min at room temperature. The plasma was then transferred to a new tube and stored frozen at -70°C. Exosome-like extracellular vesicles (ELVs) were isolated from 0.6, 1.0, and 1.7 mL plasma with or without proteinase K treatment (0, 0.5, or 1 mg/mL) using the ExoQuick Exosome Isolation Kit for Serum and Plasma, the Invitrogen Total Exosome Isolation Kit, or the miRCURY Exosome Serum/Plasma Kit. Proteins were quantified by the bicinchoninic acid (BCA) assay and levels of Alix, TSG-101, CD-63, and annexin-5 and the absence of GM130, calnexin, and apolipoprotein A-1 (apo A-1) were evaluated by Western blot. ELVs were visualized by transmission electron microscopy (TEM) and the size distribution determined by nanoparticle tracking analysis (NTA) on a Nanosight NS300. miRNA was extracted from the miRCURY extracted ELVs and from 0.5 mL plasma using the miRNeasy Serum/Plasma Advanced Ki. miRNA was reverse-transcribed using the miRCURY LNA RT Kit and levels of miR-30c, miR-126, and miR-192 were quantified by real-time RT-PCR.

   Summary of Findings:

   The highest total protein yields were with the miRCURY extraction kit. Inclusion of proteinase K resulted in decreased total protein concentration, regardless of extraction method. The highest protein yield was achieved when specimens were acidified after proteinase K treatment and then extracted using the miRCURY kit. Inclusion of a proteinase K digestion step caused a concentration-dependent decrease in the levels of all non-exosomal proteins and in the majority of exosomal proteins examined (Alix, CD63, and TSG101) but an increase in the detection of Annexin V (P<0.01). Notably, the protein concentration with and without proteinase K treatment was highly dependent on the extraction kit used with the highest levels of exosomal markers found in non-proteinase K treated specimens extracted with the ExoQuick kit. However, when treated with 0.5 or 1 mg/mL proteinase K, the levels of TSG101 and CD63 were highest using the miRCURY kit and Annexin V levels were lower than with ExoQuick. Addition of an acidification step after proteinase K treatment increased the detection of Annexin V, TSG101, and ALIX but also increased detection of the non-exosomal marker Apo-A1. The authors state the best overall detection of exosomal markers was achieved using 0.5 mg/mL proteinase K and the miRCURY kit. Importantly, addition of proteinase K decreased the presumed co-precipitates observed under TEM, regardless of extraction method, and addition of HCl did not alter the ELV morphology. The protein concentration per mL of plasma was highest when extraction was from 0.6 mL plasma but the coefficient of variance was also highest. Using NTA, it was found that 0.5 mg/mL proteinase K treatment reduced the mean size of the ELVs (188.8 versus 170.8 for ExoQuick, 171.8 versus 126.2 for Total Exosome, and 172.5 versus 125.4 for miRCURY kit). Treatment with 1 mg/mL proteinase K produced comparable NTA results to 0.5 mg/mL proteinase K when extraction was with ExoQuick or the Total Exosome kits but there were two peaks when extraction was with miRCURY. Importantly, miR-30c, miR-126, and miR-192 were detected in both the plasma and miRCURY extracted ELV specimens but only 45-65% of each found in ELVs compared to plasma.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Not specified
   • Alzheimer's Disease
   • Normal

   Platform:

   Analyte	Technology Platform
   Protein	Western blot
   Protein	Colorimetric assay
   RNA	Real-time qRT-PCR
   Morphology	Light scattering
   Morphology	Electron microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	ExoQuick Exosome Isolation Kit for Serum and Plasma kit Invitrogen Total Exosome Isolation Kit miRCURY Exosome Serum/Plasma Kit
   Biospecimen Aliquots and Components	pH	Unadjusted Low pH
   Western blot Specific	Targeted peptide/protein	TSG101 CD63 Annexin V Alix GM130 Calnexin Apo-A1
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-30c miR-126 miR-192
   Analyte Extraction and Purification	Protein digestion	Untreated 0.5 mg/mL Proteinase K 1.0 mg/mL Proteinase K
   Biospecimen Aliquots and Components	Aliquot size/volume	0.6 mL 1.0 mL 1.7 mL

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