U.S. Department of Health and Human Services

Sample Collection and Storage for Exosome Analysis

The Kidney Diseases Branch, Renal Diagnostics and Therapeutics Unit has developed a urinary sample collection, storage, and processing protocol for the isolation of urinary exosomes.  Additional information about this protocol is available in the following publication:

Gonzales, P, Zhou, H, Pisitkun, T, Star, RA, Knepper, MA, and Yuen, PST (2010) Isolation and Purification of Exosomes in Urine.  in The Urinary Proteome: Methods and Protocols, Methods in Molecular Biology 641: 89-99 A. J. Rai, ed., Humana Press, New York.

If you have questions about this protocol, please contact Dr. Peter Yuen.

Protocol

Sample collection (protease inhibitor) solution (volume per 50 ml urine)

  • 1.67 ml of 100 mM sodium azide (NaN3)
  • 2.5 ml PMSF (2 mg/ml in isopropyl alcohol, stable 4°C for several months)
  • 50 µl Leupeptin (1 mg/ml in ddH2O, stable 1 week at 4°C, 6 months at -20°C)

Collection

  • If possible, determine the collection time and volume.
  • Collect the first or second morning urine, 10~100 ml (See Notes 1 and 2).
  • First morning urine: first urine after waking (no fluid or fruits after 9 PM the prior evening).
  • Second morning urine: Discard the first urine. Collect the next voided urine. May have breakfast and undergo regular activity between first and second morning urine.
  • Can also use random urine samples

Storage

  • Add sample collection (protease inhibitor) solution (see above)
  • If possible, process samples immediately (refrigerate at 4 °C).
  • If the urine samples cannot be processed immediately, the samples should be frozen at -80 °C (NOT -20 °C). (See Note 3)
  • 50 ml of urine samples are aliquoted in 50 ml tubes and stored at -80°C as soon as possible after collection.

Defrosting and processing

  • Frozen urine samples should be thawed at room temperature (requires ~3 hrs for a 50 ml urine sample). Avoid prolonged thawing on ice (4 °C).
  • While urine is defrosting (i.e., is still a mixture of ice and water), extensively and vigorously vortex for one minute.
  • After sample completely thaws, vigorously vortex for an additional 30 sec, then proceed to differential centrifugation for urinary exosome isolation.
  • Insufficient vortexing will result in major loss of urinary exosomes.

Notes

  • 10 ml of urine is sufficient to detect exosomal TSG101, NHE3, AQP2, and ALIX by western blotting.
  • The difference between first morning urine and second morning urine is very small. Either can be used for analysis of TSG101, NHE3, AQP2, and ALIX.
  • Freezing at -20 °C causes a major loss of urinary exosomes that can be partially restored by extensive vortexing.
  • In urine from healthy individuals, 1 ml of urine yields approximately 2 ul of 200,000 × g spin pellets. The concentration of urinary exosomal protein is ~1.1 ug/ul .
  • Western blot analysis: Mix equal volume of Laemmli sample buffer (Bio-Rad) including 60 mg/ml DTT to isolation solution, heat samples at 60 °C for 10 min, then store samples at -80 °C for western blot analysis.
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