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Impact of Extracellular Vesicle Isolation Methods on Downstream Mirna Analysis in Semen: A Comparative Study.

Author(s): Mercadal M, Herrero C, López-Rodrigo O, Castells M, de la Fuente A, Vigués F, Bassas L, Larriba S

Publication: Int J Mol Sci, 2020, Vol. 21, Page

PubMed ID: 32824915 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compared small extracellular vesicle (sEV) recovery, particle size distribution, protein expression, and microRNA (miRNA, miR) levels among sEVs isolated from seminal plasma (SP) using 5 different extraction methods.  The potential influence of exosome isolation method on clinical differences in miRNA levels was also examined using SP from patients diagnosed with prostate cancer, benign prostatic hyperplasia, and healthy volunteers. Semen was collected by masturbation 3-5 days after sexual abstinence from 12 men consulting for infertility or vasectomy, 9 prostate cancer patients, and 5 patients with benign prostate hyperplasia. Semen was allowed to liquefy for 10 min at 37˚C, centrifuged at 1,600 x g for 10 min, followed by 16,000 x g for 10 min at 4˚C. Seminal plasma was aliquoted and stored at -80˚C. After storage, seminal plasma was passed through a filter before isolation of sEVs. To test the effects of extraction method, sEVs were isolated using four different kits with multiple protocols for each and ultracentrifugation. Exosomes were isolated using the miRCURY Exosome Serum/Plasma Kit using the serum protocol of no thrombin treatment, precipitation for 1 h at 4˚C, followed by centrifugation at 1500 x g for 30 min or the plasma protocol which includes thrombin treatment for 5 min at 20˚C, precipitation for 1 h at 4˚C, and centrifugation at 500 x g for 5 min. Exosomes isolated using the miRCURY Exosome Cell/Urine/CSF Kit were precipitated for 1 h at 4˚C followed by centrifugation at either 1500 or 10,000 x g for 30 min at 20˚C. Exosome isolation using the ExoQuick Ultra EV Isolation Kit for Serum and Plasma was tested using four protocols: precipitation for 30 min at 4˚C and centrifugation at 3000 x g for 10 min at 4˚C followed by purification with a column; precipitation overnight at 4˚C, and centrifugation at 1,500 x g for 10 min at 4˚C followed by purification columns; precipitation for 30 min at 4˚C and centrifugation at 3000 x g for 10 min at 4˚C; and precipitation overnight at 4˚C and centrifugation at 1500 x g for 10 min at 4˚C. Exosome isolation using the ExoGAG Kit involved precipitation for 5 min at 4˚C followed by centrifugation at 1500 or 3500 x g for 30 min at 4˚C. All methods of exosome purification were compared to ultracentrifugation at 100,000 x g for 2 h at 4˚C. All sEVs were frozen at -80˚C before analysis. The size and concentration of isolated sEVs were measured by nanoparticle tracking analysis. Levels of the markers CD81 and CD63 were analyzed by flow cytometry. sEVs were treated with RNAse and RNA was subsequently extracted using the miRNeasy Micro Kit. RNA was quantified by flourometry and purity evaluated by spectrophotometer. Levels of miRNAs were quantified by real-time PCR and normalized to the most stable miRNA (miR-576-5p and miR-181c-5p). PSA levels were determined prior to selection for the study by an unspecified method.  

   Summary of Findings:

   Fewer sEVs than the limit of detection were isolated with the miRCURY Serum/Plasma Kit using the plasma protocol (with thrombin treatment) and, while above the detection threshold, use of the serum protocol resulted in 2.8-fold fewer particles and a larger mean particle size compared to ultracentrifugation. Using the ExoQuick ULTRA Kit with purification columns yielded 266 and 25-fold fewer particles than ultracentrifugation when centrifuged at 1500 x g and 3000 x g, respectively; and the particles had a higher mean and mode size. Fewer particles were obtained using ExoGAG with either centrifugation speed than by ultracentrifugation but the particles were of a comparable size. More particles (1.5 to 2-fold) were isolated using the miRCURY Cell/Urine/CSF with centrifugation at either speed than by ultracentrifugation and the particles isolated by centrifugation at 1500 x g were of comparable size to those isolated by ultracentrifugation.

   Flow cytometry showed expression of both CD63 and CD81 on sEVs isolated using ultracentrifugation, ExoGAG with centrifugation at 3500 x g, and the miRCURY Cell/Urine/CSF Kit with centrifugation at 1500 x g, but there were differences in abundance. The intensity of CD63 was comparable in sEVs isolated by ultracentrifugation and ExoGAG with centrifugation at 3500 x g, and higher than that in sEVs purified by miRCURY Cell/Urine/CSF. CD81 intensity was higher in sEVs isolated by ultracentrifugation than ExoGAG with centrifugation at 3500 x g or using miRCURY Cell/Urine/CSF.

   RNA obtained from sEVs isolated using ExoGAG with centrifugation at 1500 or 3500 x g, the miRCURY Cell/Urine/CSF with centrifugation at 1500× g, or miRCURY Serum/Plasma Kit with the serum protocol had comparable purity 260/280 ratios to that of RNA from sEVs obtained by ultracentrifugation (1.63-1.79 versus 1.78). The RNA yield was highest using sEV isolation methods that also resulted in highest sEV yield, with the highest yield when sEVs were isolated using the miRCURY Cell/Urine/CSF Kit with centrifugation at 1500 x g (31.03 ng/µL), followed by ultracentrifugation (22.39 ng/µL) and ExoGAG with centrifugation at 3500 x g or 1500 x g (12.29 and 8.17 ng/µL, respectively), but extraction of RNA from sEVs isolated using the other methods yielded <5 ng/µL. CT values for miR-30d-5p, miR-93-5p, and miR-449a differed among RNAs isolated from sEVs obtained using the different methods, despite use of an equivalent amount of RNA.  Importantly, differences in levels of 16 miRNAs between healthy patients and those with prostate cancer depended on sEV isolation method. Levels of miR-142-3p and miR-142-5p were only diagnostically efficient to distinguish prostate cancer versus benign prostatic hyperplasia when used in combination with PSA, regardless of extraction method. A panel based on levels of miR-142-3p, miR-142-5p, and miR-223-3p was diagnostically accurate to distinguish prostate cancer versus benign prostatic hyperplasia in sEVs isolated using miRCURY Cell/Urine/CSF with centrifugation at 1500 x g (sensitivity 88.9%, specificity 80.0%, P=0.014), but not when sEV isolation was with the other methods. Addition of PSA allowed for accuracy of this panel in sEVs isolated by ultracentrifugation (sensitivity 100%, specificity 100%, P=0.009), ExoGAG (sensitivity 100%, specificity 100%, P=0.003) or miRCURY Cell/Urine/CSF with centrifugation at 1500 x g (sensitivity 100%, specificity 100%, P=0.003).

   Biospecimens

   • Bodily Fluid - Semen

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Benign
   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Light scattering
   Protein	Flow cytometry
   RNA	Spectrophotometry
   RNA	Real-time qRT-PCR
   RNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Centrifugation	Multiple speeds compared
   Real-time qRT-PCR Specific	Targeted nucleic acid	let7i-3p miR 106b-5p miR-125a-5p miR-130a-3p miR-142-5p miR-142-3p miR-181c-5p miR-196b-3P miR-223-3p miR-30c-5p miR-30e-3p miR-34a-3p miR-34a-5p miR-576-5p miR-663b miR-92a-3p miR-576+181
   Analyte Extraction and Purification	Analyte purification	Column after ExoQuick No column after ExoQuick
   Analyte Extraction and Purification	Analyte isolation method	ExoGAG Kit miRCURY Exosome Cell/Urine/CSF Kit miRCURY Exosome Serum/Plasma Kit ExoQuick ULTRA EV Isolation Kit for Serum and Plasma Ultracentrifugation

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