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Robustness of urinary extracellular vesicle-derived MiRNA profiles over multiple days and the impact of urine concentration.

Author(s): Satomura A, Ando Y, Mikami M, Mizunuma M, Ichikawa Y

Publication: Sci Rep, 2025, Vol. 15, Page 34334

PubMed ID: 41038864 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the potential effects of inter- and intra-individual variability, timing of collection (relative to food and beverage consumption, prior voids and time of day), and urine pH and concentration (volume, specific gravity, color and creatinine concentration) on the urinary cell-free miRNA profile. Urine was collected serially from six healthy volunteers (four men and two women, aged 20-40 years) over the course of three days. Volunteers self-recorded the volume of urine collected, meals (composition and time) and beverages (types and times) consumed, exercise details (type and time), medications (type and times), and times of bathing and sleeping. Urine specimens were stored at 4°C until transport (details not provided) and then stored at -80°C until analysis. Urinalysis was performed using an AUTION Sticks 10EA instrument and creatinine was quantified with the Qualigent CRE Sssay Kit.  To isolate extracellular vesicles, 3.5 mL of urine was centrifuged at 2000 g for 30 min at 4°C, the supernatant was incubated with Total Exosome Isolation Reagent for 1 h at room temperature, EVs were pelleted by centrifugation at 3000 g for 1 h at 4°C, and resuspended in phosphate buffered saline. The size and concentration of EVs were analyzed using a NanoSight LM10 system. RNA was extracted from EVs using the MagMAX mirVana Total RNA Isolation Kit and the KingFisher Apex automation system. RNA was concentrated using an Eppendorf centrifugal concentrator and stored at -80°C. Small RNA sequencing libraries were prepared using the QIAseq miRNA Library Kit and sequenced on a NextSeq 550 machine.

   Summary of Findings:

   miRNAs counts from urinary EVs were variable both within specimens from the same individual and between individuals. Generally, miRNA counts were lower during the middle of the day.  miRNA counts were strongly correlated with the percentage of miRNA reads which varied from 0.0276 to 57.7% (r=0.849). The total miRNA count was strongly correlated with the total number of miRNAs detected (r=0.984, P<0.001) and the number of miRNAs detected with >10 and >100 counts (r=0.99, P<0.001 and r=0.986, P<0.001, respectively). The variability in miRNA counts was highest in specimens with the lowest number of miRNAs counted.  miRNA counts were negatively correlated with total urine volume per day (r=-0.552, P=0.018) and with urine volume of spot voids (r=-0.562, P<0.001), but the correlation was not significant for the volume of the first void (r=-0.413, P=0.089).  miRNA counts were also negatively correlated with the volume of the urine specimen when specimens were collected within 200 min of a prior urination event. (r=-0.618, P<0.001). Interestingly, there was no correlation between the miRNA counts and the volume of caffeinated or of other fluid intake (r=0.004, P=0.976 and r-0.13, P=0.451, respectively). Not surprisingly, urine volume and fluid intake were greater during the day than at night.  miRNA counts were positively correlated with color of the urine (r=0.589, P<0.001), urine specific gravity (r=0.634, P<0.001) and urine creatinine concentration (r=0.581, P<0.001). miRNA counts were higher in specimens with a pH ≥5.5 than those with a pH of <5, and urine pH was found to be correlated with spot urine volume (r=0.233, P=0.009), creatinine (r=-0.25, P=0.032) and urine color (coefficient not included).  The urine specific gravity was correlated with creatinine (r=0.798, P<0.001) and urine color. In multiple regression analysis, urine specific gravity (P<0.001), urine color (P<0.01) and volume (P<0.05) were predictors of miRNA counts.  When all samples were included, intra-individual differences and specific gravity accounted for 33.6% and 36.8% of the variability in the miRNA profiles, respectively.  However, intra-individual differences accounted for 69.4% of the variability in the miRNA profiles, and specific gravity only accounted for 12% of the variability when specimens with low miRNA counts (<10^4.7) were excluded. Further, only when specimens with low miRNA counts were excluded did the EV miRNA profiles of the specimens from the three days of collection cluster distinctly by volunteer.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Next generation sequencing
   Small molecule	pH
   Electrolyte/Metal	Clinical chemistry/auto analyzer
   Protein	Clinical chemistry/auto analyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	pH	A range of values investigated
   Biospecimen Aliquots and Components	Aliquot size/volume	Specimen volume effects investigated
   Biospecimen Acquisition	Time of biospecimen collection	First void Spot voids Timing relative to food/drink consumption <200 min from prior void >200 min from prior void Daytime collection Night collection Collection on 3 days
   Preaquisition	Patient diet	Effect of caffeine consumption Effect of meals Effect of Other beverage consumption

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