Urinary extracellular vesicles: Assessment of pre-analytical variables and development of a quality control with focus on transcriptomic biomarker research.
Author(s): Barreiro K, Dwivedi OP, Valkonen S, Groop PH, Tuomi T, Holthofer H, Rannikko A, Yliperttula M, Siljander P, Laitinen S, Serkkola E, Af Hällström T, Forsblom C, Groop L, Puhka M
Publication: J Extracell Vesicles, 2021, Vol. 10, Page e12158
PubMed ID: 34651466 PubMed Review Paper? No
Purpose of Paper
This study investigated potential effects of storing urine specimens and isolated urinary extracellular vesicles (uEVs) in phosphate buffered saline at -20°C or -80°C on particle size, morphology, and protein expression. The authors also investigated potential differences associated with the duration of urine collection (overnight versus 24 h), storage temperature, treatment with or without protease inhibitors and/or DNAse, and inclusion of a preclearing centrifugation step on RNA yield and/or next generation sequencing (NGS) data obtained from uEVs.
Conclusion of Paper
Fresh uEVs, uEVs stored in PBS and whole urine specimens for 3, 6, 9 and 24 months at -80°C displayed comparable uEV concentrations; mean, modal, and median uEV sizes; and 10’th-90’th percentile ranges. However, uEV concentrations were lower after storage of isolated uEVs in PBS or whole urine for 14 days at -80°C compared to fresh urine. There was no effect of storing whole urine or isolated uEVs in PBS at -80°C on the SDS-PAGE protein profile; moreover, the EV markers, TSG101, HSP70, and CD9 were detected in all specimens. However, EV from urine stored at -20°C for 1.5 months had lower levels of TSG101, CD9, and CD63 and these differences were more pronounced in pooled urine from patients with diabetes that were stored for up to 4 years than in urine from healthy individuals stored for 1.5 months. uEV morphology was largely unaffected by storage of uEV in PBS or as whole urine at -80°C or storage of urine at -20°C, but particles with surface spikes that lacked CD59 expression were only identified in fresh uEV.
RNA yield was significantly lower when urine specimens were stored at -20°C than -80°C and when a DNAse treatment step was incorporated, but was not affected by use of protease inhibitors, inclusion of a preclearing centrifugation step, or collection of 24 h versus first morning urine. Percentage of mapped reads was not affected by urine storage temperature, but principal component analysis (PCA) of RNA expression clustered specimens by urine storage temperature. Compared to whole urine stored at -80°C, uEVs stored at -20°C for 1 year displayed higher expression levels of 137 genes and lower expression of 1,987 genes. While fragment length did not differ between genes with higher or lower expression, genes with higher GC content were more likely to have lower expression in uEV isolated from urine stored at -20°C. uEVs isolated from DNAse treated urine samples had a higher percentage of 3’ untranslated (UTR) region exons and a higher number of genes with >1 count in ≥50% of specimens, and a lower percentage of coding sequence exons, 5’UTR exons, introns, intragenic parts and transcription start and end sites. Mean miRNA read counts were lower in uEVs isolated from urine stored at -20°C rather than -80°C, and 4 miRNAs were found at higher levels and 29 miRNA at lower levels when urine was stored at -20°C rather than -80°C.
Studies
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Study Purpose
This study investigated potential effects of storing urine and isolated uEV in PBS at -80°C for up to 24 months on uEV particle size, morphology, and protein expression. The ability to detect viral particles in uEV isolates was also investigated. First and second morning urine from healthy patients was collected and pooled: pooled urine from 11 males, pooled urine from 7 females and 2 males, and pooled urine from 2 females and 3 males. Urine pools were aliquoted and uEVs were isolated by ultracentrifugation (8000 x g for 15 min, filtered through 1.2 µm cellulose acetate syringe filter, and centrifuged again at 100,000 x g for 90 min) immediately or after frozen storage of urine at -80°C for 14 days, 3 months, 6 months, 9 months, and 24 months. Isolated uEVs were resuspended in phosphate buffered saline (PBS) and analyzed fresh or after storage at -80°C for 14 days, 3 months, 6 months, 9 months, and 24 months. Particle concentration and size distribution were analyzed by nanoparticle tracking analysis. Morphology was evaluated by transmission electron microscopy (TEM). The protein profile of uEVs was evaluated by Coomassie blue staining of SDS-PAGE gels. Protein expression of EV specific markers, TSG101, HSP70, and CD9, were quantified by Western blot analysis. Additionally, expression of CD59 was evaluate by TEM. Viral levels of JC-polyoma and BK-polyoma in urine were quantified by real-time PCR.
Summary of Findings:
The uEV concentration, mean size, modal size, median size, and 10’th-90’th percentile range were comparable in fresh uEVs and uEVs stored in PBS or as whole urine for 3, 6, 9 and 24 months at -80°C. Interestingly, in a second study, uEV concentrations were lower after storage of uEVs in PBS (P<0.01) or whole urine for 14 days (P<0.05) compared to fresh urine, but size distributions was unaffected and there was no difference in uEV concentration after storage for 3 months. Storing whole urine or uEVs in PBS did not affect the SDS-PAGE protein profile; further, levels of the EV markers, TSG101, HSP70, and CD9 were detected in all specimens. uEV morphology was largely unaffected by storage of whole urine of uEVs in PBS at -80°C, but particles with a diameter of 56.3 ±0.1 nm with surface spikes were only identified in fresh uEVs. The spiked particles did not express the EV marker CD59. JC-polyoma and BK-polyoma were detected in uEV isolates indicating that polyoma viruses co-isolate with EVs.
Biospecimens
Preservative Types
- None (Fresh)
- Frozen
Diagnoses:
- Normal
- Diabetes Type 2
- Diabetes Type 1
Platform:
Analyte Technology Platform Cell count/volume Light scattering Cell count/volume Electron microscopy Protein 1D/2D gels Protein Western blot Morphology Electron microscopy Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 0 days
14 days
3 months
6 months
24 months
Western blot Specific Targeted peptide/protein TSG101
HSP70
CD9
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Storage Storage conditions uEV in PBS
Whole Urine
-
Study Purpose
This study investigated the potential effects of storing urine from healthy patients and those with type 1 diabetes and normo- or macroalbuminuria at -20 and -80°C on uEV morphology, protein expression, RNA yield, and RNA and miRNA sequencing data. First morning urine from 20 healthy patients and 24 h urine from 18 patients with type I diabetic kidney disease were collected for the study. Urine volume of specimens collected from patients with diabetes were classified as macroalbuminuric (9 patients, albumin excretion rate (AER)>300 mg/24 h), or normoalbuminuric (9 patients AER<30 mg/24 h) based on the 24 h urine collection specimen. Urine was stored for 1.5 months (healthy individuals), 1 year (all individuals), or 4 months - 4 years (normo- or macroalbuminuria) as 7 mL (22 specimens) or 30 mL (16 patients) aliquots. uEVs were isolated by ultracentrifugation (8000 x g for 15 min, filtered through a 1.2 µm cellulose acetate syringe filter, and centrifuged again at 100,000 x g for 90 min). Morphology was evaluated by transmission electron microscopy (TEM). Protein expression of EV-specific markers (TSG101, HSP70, CD63, podocalyxin, CD9) were quantified by Western blot analysis. RNA was isolated from the 38 patients in the first study as well as 69 additional patients (healthy, type I or type II diabetes) using the miRNeasy micro kit. RNA was quantified using pico 6000 RNA chip in Agilent 2100 Bioanalyzer. Potential effects of storage temperature on uEV transcriptome was investigated by creating sequencing libraries with the TailorMix miRNA sample prep v2 kit from specimens collected from patients with type I diabetes with normoalbuminuria, macroalbuminuria, or microalbuminuria that were stored at -20°C (4 specimens) or -80°C (16 specimens) and sequenced on a HiSeq2000. miRNA sequencing libraries were constructed using QIAseq miRNA Library Prep kit and sequenced on a NextSeq500.
Summary of Findings:
EV morphology was normal after storage of urine for 1 year at -80°C or -20°C. Compared to EVs isolated from urine stored at -80°C for 1 year, EVs from urine stored at -20°C for 1 years had less TSG101, CD9, and CD63; and, this difference was more pronounced in the pools of urine from patients with diabetes that were stored for up to 4 years than in urine from healthy individuals stored for 1.5 months. Signals for TSG101, CD9, and podocalyxin were lower in urine collected from patients with diabetes that was stored for 4 months - 4 years compared to urine from healthy controls that was stored for 1.5 months. RNA yield was significantly lower when specimens were stored at -20°C than -80°C (P<0.01), but differences were only significant in the non-albuminuria group (P<0.05). RNA yield was unaffected by storage for up to 4 years at -80°C. Further, no effect of storage duration was observed within the non-albuminuria or albuminuria (micro or macro) groups, when investigated in specimens from males only, or when broken down by tube volume. Storage of urine at -20°C rather than -80°C resulted in a slightly lower average number of raw reads (4.5E+06 versus 5.3E+06), but the difference was not significant and there was no difference in the percentage mapped reads. However, PCA specimens clustered by storage temperature. Compared to urine stored at -80°C, those stored at -20°C had higher levels of 137 genes and lower levels of 1987 and transcripts with higher levels were more likely to be associated with P-bodies than down-regulated transcripts (49% versus 10%). There was no difference in fragment length between up and down-regulated genes, but genes with higher GC content were more likely to be display a reduction in transcript levels after storage at -20°C compared to specimens stored at -80°C. Further investigation revealed that urine stored at -20°C rather than -80°C had a higher percentage of genes with GC content <50% (81.4% versus 71.6%, P<0.01) and lower percentages of genes with a GC content of 51-60% (15.9% versus 23.6%, P<0.01) and 61-80% (4.7% versus 2.6%, P<0.01). Pathways analysis showed genes with lower expression after storage at 20°C compared to -80°C were associated with carbohydrate and lipid metabolism. Mean miRNA read counts were lower in urine stored at -20°C rather than -80°C (4.57E+ 04 versus 8.7E + 05, P<0.05), and 4 miRNAs were found at higher levels and 29 miRNAs at lower levels when urine was stored at -20°C rather than -80°C.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
- Diabetes Type 1
Platform:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer Morphology Electron microscopy Protein Western blot RNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Aliquot size/volume 30 mL urine
7.5 mL urine
Preaquisition Diagnosis/ patient condition Healthy
Type I diabetes with normoalbuminuria
Type I diabetes with macroalbuminuria
Storage Storage duration 1.5 months
4 months-4 years
Storage Storage temperature -20°C
-80°C
Western blot Specific Targeted peptide/protein HSP70
TSG101
CD63
Podocalyxin
CD9
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Study Purpose
The purpose of this study was to investigate potential differences in RNA yield and NGS data between first morning urine and 24 h urine collections; and including versus omitting treatment with protease inhibitors or DNAse, and a preclearing centrifugation. Potential effects of protease inhibitor treatment were investigated by comparing RNA yields from 17 overnight urine specimens from healthy patients treated with protease inhibitors before frozen storage at -80°C for <14 days or 24 months to first morning urine from 6 healthy patients and 4 pools of first morning urine specimens from healthy patients that were left untreated before storage at -80°C for <14 days or 24 months. The effects of urine collection method (first morning versus 24 h) on RNA yield was investigated by comparing yield from matched first morning urine and 24 h urine specimens collected from 4 healthy males, 4 males with type I diabetes and microalbuminuria and 4 males with type I diabetes and macroalbuminuria after storage at -80°C. The effect of preclearing on RNA yield was investigated by preclearing (centrifugation at 1800 x g for 10 min) half of each urine specimen from 2 males with type I diabetes and microalbuminuria and 2 males with type I diabetes and macroalbuminuria before freezing at -80°C. The effect of DNAse treatment was investigated by collecting first morning urine from each of 6 healthy volunteers on two days and treating one with DNAse while the other remained untreated. uEVs were isolated by ultracentrifugation (8000 x g for 15 min, filtered through 1.2 µm cellulose acetate syringe filter, and centrifuged again at 100,000 x g for 90 min). RNA was isolated using the miRNeasy micro Kit, with the exception of the DNase study where RNA was extracted with the Macherey-Nagel Nucleospin miRNA kit (2 patients) or the miRNeasy mini kit (3 patients). RNA was quantified using pico 6000 RNA chip with an Agilent 2100 Bioanalyzer. The Potential effects of DNAse treatment on uEV transcriptome was investigated by creating sequencing libraries with the TailorMix miRNA Sample Prep v2 Kit from 19 case-matched specimens that were DNAse-treated and those that remained untreated.
Summary of Findings:
RNA yields were not significantly different among specimens stored at -80°C with or without protease inhibitors after <14 days or 24 months. RNA yields were comparable between 24 h collections and overnight urine specimens collected from the same men. Similarly, performing a preclearing centrifugation step before freezing had no impact on RNA yield. In contrast, specimens that did not undergo an in column DNAse treatment had 20% more RNA and 23% more DNA than those that underwent DNAse treatment, regardless of RNA isolation method. DNAse-treated urine samples had a higher percentage of 3’ untranslated (UTR) region exons and a greater number of genes with >1 count in ≥50% of specimens, but a lower percentage of coding sequence exons, 5’UTR exons, introns, intragenic parts and transcription start and end sites than untreated case-matched urine specimens.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
- Diabetes Type 1
Platform:
Analyte Technology Platform RNA Next generation sequencing RNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Protease inhibitor Added before storage
No protease inhibitor added
Analyte Extraction and Purification Nucleic acid digestion DNAse treated
Not DNAse treated
Biospecimen Aliquots and Components Centrifugation Centrifuged
Not centrifuged
Biospecimen Acquisition Method of fluid acquisition Voided urine (24-h collection)
Voided urine (overnight collection)
Storage Storage conditions Precleared
Not precleared