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

Urine features an ideal source of non-invasive diagnostic markers. Some intrinsic and methodological issues still pose barriers to its full potential as liquid biopsy substrate. Unlike blood, urine concentration varies with nutrition, hydration and environmental factors. Urine is enriched with EVs from urinary-genital tract, while its conservation, purification and normalization can introduce bias in analysis of EV subsets in inter-and intra-individual comparisons.

Researchers at IRCCS San Raffaele Scientific Institute have evaluated the methods that decrease such biases such as appropriate and feasible urine storage, optimal single-step EV purification method for recovery of proteins and RNAs from small urine volumes and a normalization method for quantitative analysis of urine EV RNAs. Ultracentrifugation, chemical precipitation and immuno-affinity were used to isolate EVs from healthy donors’ urine that was stored frozen or at room temperature for up to 6 months. Multiple urine biochemical and EV parameters, including particle count and protein content, were compared across urine samples. To this purpose nanoparticle tracking analysis (NTA) and protein assessment by BCA, ELISA and WB assays were performed. These measurements were correlated with relative abundances of selected EV mRNAs and miRNAs assessed by RT-PCR and ranked for the ability to reflect and correct for EV content variations in longitudinal urine samples. All purification methods enabled recovery and downstream analysis of EVs from as few as 1 ml of urine.

These findings highlight long term stability of EV RNAs upon urine storage at RT as well as excellent correlation of EV content in urine with some routinely measured biochemical features, such as total urine protein and albumin, but not creatinine most conventionally used for urine normalization. Comparative evaluation of mRNA and miRNAs in EV isolates revealed specific RNAs, in particular RNY4 and small miRNA panel, levels of which well reflected the inter-sample EV variation and therefore useful as possible post-analytical normalizers of EV RNA content. The researchers describe some realistic urine processing and normalization solutions for unbiased readout of EV biomarker studies and routine clinical sampling and diagnostics providing the input for design of larger validation studies employing urine EVs as biomarkers for particular conditions and diseases.

Quantification of EV RNA and MV RNA/miRNA content

Figure 4

Total RNA is extracted from MV pellets and from EVs purified from healthy donor-obtained urine samples (N = 3), stored either pre-served at room temperature (P) or frozen (F), using ultracentrifugation-based protocol (UC), chemical precipitation (CP) or immunoprecipitation with anti-CD9 coated beads (IP). RT-qPCR amplification of mRNAs prior described to be contained in urine EVs, namely β-actin and RNY RNA was done as de-scribed in Material and Methods. Both these mRNAs and miRNAs (miR-16, miR-21, miR-210 and miR.451) were analyzed also in MVs. Samples from three healthy donors, and 2 independently processed aliquots from each donor, are included in the assessment. One-way ANOVA and Bonferroni post-hoc test were used to determine statistical significance of observed differences * indicates p-value < 0.05, ** p-value < 0.01 and *** p-value < 0.001.

Vago R, Radano G, Zocco D, Zarovni N. (2022) Urine stabilization and normalization strategies favor unbiased analysis of urinary EV content. Sci Rep 12(1):17663. [article]

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