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A simplified method to recover urinary vesicles for clinical applications, and sample banking.

Author(s): Musante L, Tataruch D, Gu D, Benito-Martin A, Calzaferri G, Aherne S, Holthofer H

Publication: Sci Rep, 2014, Vol. 4, Page 7532

PubMed ID: 25532487 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared urinary exosome isolation by HFD with isolation by differential centrifugation. Urine from 4 healthy individuals was centrifuged at 2000 x g and the supernatant was subjected to Hydrostatic Filtration Dialysis to produce a specimen retained above 1,000 kDa MWCO (HFDa) and the flow-through (HFDb). The retained specimen was centrifuged at 40,000 x g producing a supernatant and pellet. The supernatant was centrifuged again at 200,000 x g producing a supernatant and pellet. An additional 2 L of pooled urine specimens was split and processed by differential centrifugation (centrifugation at 2,000 x g, 17,000 x g, pellets combined with DTT, ultracentrifuged at 200,000 x g)  or using the hydrostatic filtration dialysis protocol above.

   Summary of Findings:

   Exosomes were retained in the HFD filter as determined by immunoblotting for the exosomal marker tumour suppressor gene 101 (TSG101), apoptosis-linked gene 2-interacting protein X (ALIX), dipeptidyl dipeptidase 4 (DPP4), neprilysin (NEP) and podocin and albumin was detected in the flow-through. Ultracentrifugation after HFD resulted in loss of some signal and TEM confirmed that both the 40,000 and 200,000 x g pellets contained vesicles of the correct size and morphology to be exosomes. Importantly, HFD resulted in higher protein yields and higher yields of the exosomal protein TSG101, regardless of urine volume, compared to the differential centrifugation protocol. In the differential centrifugation protocol, exosomes were in each step following the 17,000 x g centrifugation but later could be separated by HFD. Importantly, ultracentrifugation failed to recover all of the exosomes. Differential centrifugation of the HFD product showed that the majority, but not all vesicles, sediment at 5000-40,000 x g. Further, the initial centrifugation removed 66.9% of the Tamm-Horsfall Protein (THP) which is known to interfere with protein quantification was removed by and the remainder of THP was present in both HFD fractions. RNA located in the HFD product was enriched below 1000 nt and lacked ribosomal RNA.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Protein	Western blot
   Morphology	Electron microscopy
   RNA	Automated electrophoresis/Bioanalyzer
   Protein	1D/2D gels

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Western blot Specific	Targeted peptide/protein	TSG101 ALIX DPP4 NEP podocin Albumin THP
   Analyte Extraction and Purification	Analyte isolation method	Differential centrifugation Hydration filtration dialysis

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