Limits...
Different EV enrichment methods suitable for clinical settings yield different subpopulations of urinary extracellular vesicles from human samples.

Royo F, Zuñiga-Garcia P, Sanchez-Mosquera P, Egia A, Perez A, Loizaga A, Arceo R, Lacasa I, Rabade A, Arrieta E, Bilbao R, Unda M, Carracedo A, Falcon-Perez JM - J Extracell Vesicles (2016)

Bottom Line: We compared the results of the differential ultracentrifugation procedure with 4 of these methods.In our conditions, the extraction with Norgen's reagent achieved the best performance in terms of gene transcript and protein detection and reproducibility.Taken together, our results show that the isolation of uEVs is feasible from small volumes of urine and avoiding ultracentrifugation, making easier the analysis in a clinical facility.

View Article: PubMed Central - PubMed

Affiliation: CIC bioGUNE, Bizkaia Technology Park, Derio, Spain.

ABSTRACT
Urine sample analysis is irreplaceable as a non-invasive method for disease diagnosis and follow-up. However, in urine samples, non-degraded protein and RNA may be only found in urinary extracellular vesicles (uEVs). In recent years, various methods of uEV enrichment using low volumes of urine and unsophisticated equipment have been developed, with variable success. We compared the results of the differential ultracentrifugation procedure with 4 of these methods. The methods tested were a lectin-based purification, Exoquick (System Biosciences), Total Exosome Isolation from Invitrogen and an in-house modified procedure employing the Exosomal RNA Kit from Norgen Biotek Corp. The analysis of selected gene transcripts and protein markers of extracellular vesicles (EVs) revealed that each method isolates a different mixture of uEV protein markers. In our conditions, the extraction with Norgen's reagent achieved the best performance in terms of gene transcript and protein detection and reproducibility. By using this method, we were able to detect alterations of EVs protein markers in urine samples from prostate cancer adenoma patients. Taken together, our results show that the isolation of uEVs is feasible from small volumes of urine and avoiding ultracentrifugation, making easier the analysis in a clinical facility. However, caution should be taken in the selection of the enrichment method since they have a differential affinity for protein uEVs markers and by extension for different subpopulation of EVs.

No MeSH data available.


Related in: MedlinePlus

Correlation analysis for uEV proteins from BPH and PCA patients. A correlation matrix was constructed using absolute densitometry values (background subtracted) for each protein. To avoid the loss of data, a value of 0 was given to non-detected proteins. The numbers correspond to r coefficients, and only significant values (p<0.05) are coloured using a proportional colour r-scale (for PCA, n=18 and for BPH, n=9). The values employed to generate the correlation matrix can be found in Supplementary Table XI.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4759834&req=5

Figure 0006: Correlation analysis for uEV proteins from BPH and PCA patients. A correlation matrix was constructed using absolute densitometry values (background subtracted) for each protein. To avoid the loss of data, a value of 0 was given to non-detected proteins. The numbers correspond to r coefficients, and only significant values (p<0.05) are coloured using a proportional colour r-scale (for PCA, n=18 and for BPH, n=9). The values employed to generate the correlation matrix can be found in Supplementary Table XI.

Mentions: Correlation matrices and associated p-values were obtained using cor function and plotted using corrplot package of R v3.1.0 programme (2014-04-10). P-values lower than 0.05 were considered significant. Specifically, we assessed each correlation as follows: In Fig. 4, we correlated the Ct values for each gene and sample (n=10).A value of 45 was assigned to non-detected genes to avoid the loss of data. In Fig. 6, we present, for each group of patients (PCA=18, BPH=9), the correlation between the values obtained by the densitometry analysis of the Western blotting for the different proteins in each sample. In Supplementary Fig. 4, we correlate the values obtained by the densitometry analysis of the Western blotting for the different proteins in each sample. We conducted a separate correlation analysis for each isolation method (n=10). In Supplementary Fig. 8b, we correlated the Ct values obtained by qPCR for the different gene transcripts within each sample. We prepared a correlation matrix for each group of patients (PCA=18, BPH=9). The data values for each correlation matrix are provided in Supplementary Tables as indicated in the legend of each figure.


Different EV enrichment methods suitable for clinical settings yield different subpopulations of urinary extracellular vesicles from human samples.

Royo F, Zuñiga-Garcia P, Sanchez-Mosquera P, Egia A, Perez A, Loizaga A, Arceo R, Lacasa I, Rabade A, Arrieta E, Bilbao R, Unda M, Carracedo A, Falcon-Perez JM - J Extracell Vesicles (2016)

Correlation analysis for uEV proteins from BPH and PCA patients. A correlation matrix was constructed using absolute densitometry values (background subtracted) for each protein. To avoid the loss of data, a value of 0 was given to non-detected proteins. The numbers correspond to r coefficients, and only significant values (p<0.05) are coloured using a proportional colour r-scale (for PCA, n=18 and for BPH, n=9). The values employed to generate the correlation matrix can be found in Supplementary Table XI.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4759834&req=5

Figure 0006: Correlation analysis for uEV proteins from BPH and PCA patients. A correlation matrix was constructed using absolute densitometry values (background subtracted) for each protein. To avoid the loss of data, a value of 0 was given to non-detected proteins. The numbers correspond to r coefficients, and only significant values (p<0.05) are coloured using a proportional colour r-scale (for PCA, n=18 and for BPH, n=9). The values employed to generate the correlation matrix can be found in Supplementary Table XI.
Mentions: Correlation matrices and associated p-values were obtained using cor function and plotted using corrplot package of R v3.1.0 programme (2014-04-10). P-values lower than 0.05 were considered significant. Specifically, we assessed each correlation as follows: In Fig. 4, we correlated the Ct values for each gene and sample (n=10).A value of 45 was assigned to non-detected genes to avoid the loss of data. In Fig. 6, we present, for each group of patients (PCA=18, BPH=9), the correlation between the values obtained by the densitometry analysis of the Western blotting for the different proteins in each sample. In Supplementary Fig. 4, we correlate the values obtained by the densitometry analysis of the Western blotting for the different proteins in each sample. We conducted a separate correlation analysis for each isolation method (n=10). In Supplementary Fig. 8b, we correlated the Ct values obtained by qPCR for the different gene transcripts within each sample. We prepared a correlation matrix for each group of patients (PCA=18, BPH=9). The data values for each correlation matrix are provided in Supplementary Tables as indicated in the legend of each figure.

Bottom Line: We compared the results of the differential ultracentrifugation procedure with 4 of these methods.In our conditions, the extraction with Norgen's reagent achieved the best performance in terms of gene transcript and protein detection and reproducibility.Taken together, our results show that the isolation of uEVs is feasible from small volumes of urine and avoiding ultracentrifugation, making easier the analysis in a clinical facility.

View Article: PubMed Central - PubMed

Affiliation: CIC bioGUNE, Bizkaia Technology Park, Derio, Spain.

ABSTRACT
Urine sample analysis is irreplaceable as a non-invasive method for disease diagnosis and follow-up. However, in urine samples, non-degraded protein and RNA may be only found in urinary extracellular vesicles (uEVs). In recent years, various methods of uEV enrichment using low volumes of urine and unsophisticated equipment have been developed, with variable success. We compared the results of the differential ultracentrifugation procedure with 4 of these methods. The methods tested were a lectin-based purification, Exoquick (System Biosciences), Total Exosome Isolation from Invitrogen and an in-house modified procedure employing the Exosomal RNA Kit from Norgen Biotek Corp. The analysis of selected gene transcripts and protein markers of extracellular vesicles (EVs) revealed that each method isolates a different mixture of uEV protein markers. In our conditions, the extraction with Norgen's reagent achieved the best performance in terms of gene transcript and protein detection and reproducibility. By using this method, we were able to detect alterations of EVs protein markers in urine samples from prostate cancer adenoma patients. Taken together, our results show that the isolation of uEVs is feasible from small volumes of urine and avoiding ultracentrifugation, making easier the analysis in a clinical facility. However, caution should be taken in the selection of the enrichment method since they have a differential affinity for protein uEVs markers and by extension for different subpopulation of EVs.

No MeSH data available.


Related in: MedlinePlus