Table 4.
Study | Urine source | Urine sample collection | uEV isolation* (force, duration and temperature) | Isolation of total RNA or uEV RNA? | uEV nucleic acid analysis |
---|---|---|---|---|---|
Miranda et al. (2010)78 | Healthy | Timed (24 h) urine collection |
|
|
Targeted real-time PCR of mRNA for podocyte proteins |
Alvarez et al. (2012)70 | Healthy | Spot urine (1st morning void) |
|
AllPrep DNA/RNA/Protein Mini kit and RNeasy MinElute Cleanup kit (Qiagen) | Targeted real-time PCR for miRNA and mRNA of renal proteins |
Cheng et al. (2013)95 | Healthy | Spot urine (1st morning void) |
|
|
Deep sequencing of RNA |
Barutta et al. (2013)149 | Healthy, DN | Timed (overnight) urine collection | UC (200,000 g, 75 min, 4 °C) | Total RNA isolation using Trizol reagent (Invitrogen) | Targeted miRNA analysis using real-time PCR |
Lv et al. (2014)147 | Healthy, DN, FSGS, IgA | Spot urine (1st morning void) | UC (2 h, temperature not specified) | uEV-RNeasy kit (Qiagen) | Targeted real-time PCR of mRNA for podocyte proteins |
Chen et al. (2014)145 | Healthy, AKI | Spot urine | UC (200,000 g, 1 h, 4 °C) | uEV-QiAMP kit (Qiagen) | Targeted real-time PCR of mRNA for podocyte proteins |
Channavajjhala et al. (2014)85 | Healthy | Spot urine (1st morning void) adjusted to pH 7 and protease inhibitors added |
|
|
Targeted real-time PCR of mRNA for specific miRNA |
Murakami et al. (2014)144 | Healthy | Timed (12 h) urine collection |
|
Proprietary N-lauroylsarcosine-based lysis buffer | Targeted real-time PCR of mRNA for renal proteins |
Ichii et al. (2014)148 | Healthy, LN | Spot urine | UC (75 min, 4 °C) | Total RNA isolation using miRNeasy Mini kit (Qiagen) | Targeted miRNA using qPCR |
Eissa et al. (2016)150 | Type 2 diabetes | Spot urine | Direct analysis of exosome miRNA by extraction | Urine Exosome RNA Isolation Kit (Norgen Biotek) | Targeted miRNA using qPCR |
Khurana et al. (2017)172 | Healthy, CKD | Spot urine | UC (187,000 g, 90 min, 25 °C) | Total RNA isolation using TRI Reagent (Sigma) | Deep sequencing of RNA |
Gracia et al. (2017)168 | Healthy | Spot urine (2nd morning void) | UC (235,000 g, 135 min, 4 °C) | miRNeasy Mini kit with RNeasy MinElute Cleanup Kit (Qiagen) | RNA-seq, targeted miRNA qPCR and targeted protein immunoblotting |
AKI, acute kidney injury; CKD, chronic kidney disease; DN, diabetic nephropathy; DTT, dithiothreitol; FSGS, focal segmental glomerulosclerosis; IgA, IgA nephropathy; LN, lupus nephritis; miRNA, microRNA; MWCO, molecular weight cut-off; P1, procedure 1; qPCR, quantitative real-time PCR; UC, ultracentrifugation; uEV, urinary extracellular vesicle; UF, ultrafiltration.
Urine precleared of large particulate or flocculate material, usually by two sequential centrifugation steps, a short (5–30 min), low speed (1,500–3,000g) step and a longer (20–60 min), higher speed (14,000–17,500g) step.