Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Urine stabilization and normalization strategies favor unbiased analysis of urinary EV content.

Author(s): Vago R, Radano G, Zocco D, Zarovni N

Publication: Sci Rep, 2022, Vol. 12, Page 17663

PubMed ID: 36271135 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare particle concentration, size, protein content, mRNA content and miRNA levels in EVs isolated by different methods; isolated after urine storage at room temperature with a preservative or at -20°C; and after frozen storage of urine; at -80°C for 1 and 6 months. First morning urine was collected from three healthy Caucasian males (35-45 years of age). Urine was stored for <1 h at room temperature before centrifugation at 300 g for 15 min after which urine aliquots were supplemented with Norgen Urine Preservative and stored at room temperature or frozen at -20°C; urine was then stored at -80°C for 1 month or 6 months until EV isolation. Frozen specimens were thawed at 37°C and centrifuged at 300 g prior to analysis.  Micro vessicles were removed from urine by centrifugation at 12,000 g and EVs were isolated by either ultracentrifugation (15000 g for 10 min and 110,000 g for 2 h), chemical precipitation using the Exo- Prep precipitation reagent, or immunoprecipitation using anti-CD9 coated H2-latex Beads. RNA was isolated from EVs using a HansaBiomed RNA Isolation Kit and levels of B-actin, prostate specific antigen (PSA), and ENY4 were quantified by real-time PCR. EV particles were analyzed by nanoparticle analysis and protein content was evaluated by bicinchoninic acid assay (BCA) and ELISA using antibodies to CD9 and CD63.

   Summary of Findings:

   Total particle concentration and particle size were comparable between frozen and preserved specimens of each type (precleared urine, urine MV pellets, EVs isolated by ultracentrifugation and EVs isolated by chemical precipitation); however, particle concentration depended on specimen type and EV isolation method, as particle concentration was highest in EVs isolated by ultracentrifugation and lowest in EVs isolated by chemical precipitation. Protein concentrations were highest in MV pellets followed by precleared urine. Interestingly, particle concentration was generally not significantly affected by storage, but was slightly reduced when urine was stored at room temperature with a preservative for 6 months  compared to when specimens were stored at -80°C. In contrast, when stored for 1 month, particle numbers were slightly (but not significantly) higher when urine was stored at room temperature with a preservative than when stored at -80°C. When stored for 1 month, protein concentration was higher in EVs isolated by ultracentrifugation from urine stored at room temperature with a preservative than urine stored at -80°C.  However, protein content was higher in ultracentrifugation EVs isolated from urine stored frozen at -80°C for 6 months than those isolated from urine stored at room temperature.  Protein co-expression of CD9 and CD63 by ELISA were higher among EVs isolated from urine stored for 1 month at room temperature with a preservative by either ultracentrifugation or chemical precipitation than when urine stored for 1 month was used or when urine stored frozen (at-80°C) was used Western blot bands for ALIS and Tsg101 were more intense when EVs were isolated from urine stored frozen for 6 months compared to storage at room temperature with a preservative, but CD63 and CD9 bands were more intense among specimens stored at room temperature. The authors attributed this increase in CD63 and CD9 intensity to compromised vesicle integrity after room temperature storage. While urine RNA concentrations were Insufficient for quantification in all specimens, miRNA contents were dependent on storage and EV isolation method. EVs isolated by ultracentrifugation from urine stored at room temperature with a preservative as opposed to frozen at -80°C had higher levels of miR-16, miR-21, miR-210, and miR-451 after 1 month (P<0.001, P<0.001, P<0.05 and P<0.01, respectively), higher levels of miR-21 and miR-451 after 6 months (P<0.01, and P<0.05, respectively), and lower levels  of miR-16 after 6 months (P<0.05).  Comparatively, EVs isolated by chemical precipitation from urine stored at room temperature with a preservative as opposed to frozen at -80°C had higher levels of miR-21 and miR-451 after 1 month (P<0.001 and P<0.01, respectively), lower levels of miR-210 after 1 month (P<0.01), higher levels of miR-21 after 6 months (P<0.01), and lower levels of miR-16 after 6 months (P<0.001). EVs isolated by immunoprecipitation from urine stored at room temperature with a preservative as opposed to urine that was frozen at -80°C had higher levels of miR-210 after 1 month (P<0.01), lower levels of miR-16 and miR-21 after 1 month (P<0.05and P<0.01, respectively), higher levels of miR-210 after 6 months (P<0.05), and lower levels of miR-21 after 6 months (P<0.01). The concentration of B-actin mRNA was higher in EVs isolated from urine stored at room temperature with a preservative than urine that was stored frozen when isolation was by ultracentrifugation or immunoprecipitation (P<0.01, both).

    

    

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Other Preservative
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Light scattering
   Protein	ELISA
   Protein	Western blot
   RNA	Fluorometry
   RNA	Real-time qRT-PCR
   RNA	Automated electrophoresis/Bioanalyzer
   Protein	Colorimetric assay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Frozen Norgen urine preservative
   Storage	Storage temperature	-80°C Room temperature
   Storage	Storage duration	1 month 6 months
   Analyte Extraction and Purification	Analyte isolation method	Differential ultracentrifugation Immunoprecipitation Chemical precipitation

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2. Study Purpose

   The purpose of this study was to assess variability in particle count and protein, mRNA, and miRNA content. This study also identified methods to reduce variability using urine specimens collected from two patients on three consecutive days (one male and one female volunteer). First morning urine was collected from three healthy Caucasian males (35-45 years of age). Urine was stored for <1 h at room temperature before centrifugation at 300 g for 15 min. Microvessicles (MVs) were isolated and removed from urine by centrifugation at 12,000 g and extracellular vesicles (EVs) were isolated by ultracentrifugation (15000 g for 10 min and 110,000 g for 2 h). RNA was isolated from EVs using a HansaBiomed RNA Isolation Kit and levels of B-actin, PSA and ENY4 were quantified by real-time PCR. EV particles were analyzed by nanoparticle analysis and protein content was evaluated by BCA and ELISA using antibodies against CD9 and CD63. miRNA levels were quantified using TaqMan real-time PCR cards.

   Summary of Findings:

   Protein content (as measured by BCA and Western blot) and particle concentration were highly variable among longitudinal collections in precleared urine, and in microvesicle (MV) pellets and extracellular vesicles (EVs) that were isolated by ultracentrifugation. Whole urine particle counts were not correlated with particle counts in EVs, but protein content was correlated between precleared urine and EV specimens. Of the proteins examined, CD9 expression was most representative of EV particle number and total protein content, with stronger correlations observed between CD9 expression and particle number in urine (R = 0.860) than in EV pellets (R = 0.607).  EV protein content and particle concentration were strongly correlated in both isolated EVs (R = 0.781) and in whole urine (R = 0.817).  Of the biochemical parameters assessed, urine total protein content and urine albumin level were most strongly correlated with total particle counts (R = 0.875 and R=0.961, respectively), EV protein level (R = 0.980 and R=0.907, respectively), and CD9 expression (R = 0.937 and R=0.922, respectively). The authors found that normalization of protein concentration, particle count, and CD9 intensity to total protein content resulted in low intra- and inter-individual variability. Protein levels of B-Actin, PSA, and RNY4 were variable between consecutive collection days for both extracted EVs and MVs. RNY4 levels were strongly correlated with particle counts (R = 0.987), protein content (R = 0.0777), CD9 expression (R = 0.837) and albumin content (R = 0.900), but only modestly correlated to creatinine (R = 0.585).  However, correction of RNY4 levels with total protein content did not decrease inter- or intra-sample variability, but correction of RNY4 levels with CD9 expression did decrease inter-sample variation to <10%.  When levels of 24 miRNA were evaluated, the authors identified two clusters: miRNAs that were uniformly expressed regardless of EV concentration, and miRNAs that varied across longitudinal samples but were correlated with particle counts (R=0.947-0.986), CD9 expression (R=0.644-0.920), and albumin levels (R=0.886-0.943). The inter-specimen variability in miRNA levels was reduced when expression was normalized to particle count or CD9 expression.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Light scattering
   Protein	Colorimetric assay
   RNA	Low density array
   Protein	Western blot
   RNA	Real-time qRT-PCR
   Protein	ELISA

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Biospecimen heterogeneity	Multiple specimens analyzed
   Biospecimen Aliquots and Components	Biospecimen components	Whole urine EVs MVs
   Real-time qRT-PCR Specific	Data handling	Normalization to CD9 Normalization to particle count Normalization to albumin Normalization to total protein
   Biospecimen Aliquots and Components	Aliquot sequential collection	Serial collections compared
   Biospecimen Acquisition	Time of biospecimen collection	Collected on three consecutive days

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