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. Author manuscript; available in PMC: 2018 May 9.
Published in final edited form as: Nat Rev Nephrol. 2017 Oct 30;13(12):731–749. doi: 10.1038/nrneph.2017.148

Table 4.

Transcriptomic analysis of extracellular vesicles in human urine

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

  • P1: UC (118,000 g, 70 min, 4 °C)

  • P2: UF (100 kDa MWCO, 4,000 g, 4 min, 25 °C)

  • P3: Percoll density gradient centrifugation (50,000 g, 45 min, temperature not specified)

  • P1: total RNA using ZR Urine RNA Isolation kit (Zymo Research)

  • P2: uEV RNA using RNeasy Plus Micro kit (Qiagen)

  • P3: uEV RNA using RNeasy Plus Micro kit (Qiagen)

 Targeted real-time PCR of mRNA for podocyte proteins
Alvarez et al. (2012)70 Healthy Spot urine (1st morning void)

  • P1: UC (165,000 g, 70 min, 37 °C)

  • P2: UC (165,000 g, 70 min, 37 °C), followed by UC of pellet through 30% sucrose cushion

  • P3: pre-filter through 0.22 μm filter, then UC (165,000 g, 75 min, 37 °C)

  • P4: UF (VivaSpin 20, 3,000 g, 60 min, temperature not specified)

  • P5: ExoQuick-TC (12 h, 4 °C) with centrifugation (1,500 g, 30 min, 25 °C)

  • P6: ExoQuick-TC plus 1.3 M DTT (12 h, 4 °C) with centrifugation (10,000 g, 30 min, 25 °C)

 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)

  • P1: UC (200,000 g, 65 min, 4 °C)

  • P2: UC (200,000 g, 65 min, 4 °C); 37 °C incubation with 200 mg/ml DTT for 10 min and UC (65 min, 4 °C)

  • P1: miRNeasy kit (Qiagen)

  • P2: miRNeasy with RNeasy MinElute Cleanup kit (Qiagen)

  • P3: mirVana PARIS kit (Ambion)

  • P4: Trizol LS reagent (Life Technologies)

  • P5: miRCURY RNA isolation kit (Exiqon)

  • P6: Urine Exosome RNA isolation kit (Norgen Biotek)

 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

  • P1: UF (Vivaspin 20, 100 kDa MWCO membranes, 4 °C)

  • P2: UC (1 h)

  • P3: precipitation using ExoQuick-TC; overnight incubation in ~9% ExoQuick

  • P1: TRI Reagent (Life Technologies)

  • P2: TRI Reagent plus miRNeasy kit

  • P3: miRNeasy kit

  • P4: mirVana miRNA isolation kit

  • P5: miRCURY RNA isolation kit

  • P6: SeraMir exoRNA columns

 Targeted real-time PCR of mRNA for specific miRNA
Murakami et al. (2014)144 Healthy Timed (12 h) urine collection

  • P1: exosome collection tube (Hitachi Chemical Research Center, Irvine, CA)

  • P2: UC (100,000 g, 1 h, unknown temperature)

 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.