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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

Western blot analysis of 2 representative independent biological samples. uEVs were enriched using indicated methodologies, in duplicate. uEV-enriched preparations were analysed by Western blot using antibodies against indicated proteins. Molecular weights are shown.
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Figure 0002: Western blot analysis of 2 representative independent biological samples. uEVs were enriched using indicated methodologies, in duplicate. uEV-enriched preparations were analysed by Western blot using antibodies against indicated proteins. Molecular weights are shown.

Mentions: Next, we evaluated several EV protein markers including CD9, CD10, CD63, TSG101, CD10, AIP1/Alix, AQP2 and FLT1. In Fig. 2, we show the WB analysis of these proteins in 2 independent biological samples (each of them in duplicated) isolated by using the 5 different methods. We found that the different methods yielded different amounts of these EV protein markers. We also noted that the electrophoretic protein mobilities obtained using INV methodology differed slightly from the protein mobilities observed for the other methods (Fig. 2). In addition, we observed clear differences between individuals, in accord with the interindividual variability shown in several studies of EVs in human biofluids. For instance, sample M3 had weaker AIP1 and CD9 bands but stronger CD10, AQP2 and CD26 bands than sample F3. To obtain quantitative data, densitometry and background subtraction for the Western blotting bands of the 10 independent samples were performed (see values in Supplementary Table III). The absolute values were transformed into relative values by referring to the highest value obtained for each protein and for each sample (Fig. 3a). For instance, NOR methodology gives a value of 100% for AIP1 in the samples shown in Fig. 2. For CD63, the 100% value was given to the levels obtained using CEN methodology. Figure 3a shows the average of the quantitative results for all samples. It was clear that NOR method resulted in a mixture enriched in AIP1 and CD26 but with lower levels of AQP2 and CD63. CEN sediments vesicles enriched in CD63, CD9 and AQP2, with lower levels of vesicles containing AIP1 (Fig. 3a). Both methods (NOR and CEN) isolated a mixture containing CD10 and FLT1, with similar efficiency. LEC-based method seemed to be very specific for CD9-positive vesicles and had the lowest efficiency for the isolation of AIP1 and CD26-containing vesicles (Fig. 3a). In contrast, INV-based methodology had high efficiency for obtaining CD26 and CD63 proteins (Fig. 3a). Remarkably, under the conditions used, EXQ method was the least efficient for most of the uEV protein markers (CD63 was detected at a relatively high level, Fig. 3a). To discard for the presence of uEVs markers as soluble proteins, we performed Western blot analysis loading NOR and CEN methodologies along with 15 µl of urine sample directly (Supplementary Fig. 3). The results showed that no detection was observed in urine sample loaded directly and uEVs protein markers were only detected when the isolation procedures were performed.


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)

Western blot analysis of 2 representative independent biological samples. uEVs were enriched using indicated methodologies, in duplicate. uEV-enriched preparations were analysed by Western blot using antibodies against indicated proteins. Molecular weights are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 0002: Western blot analysis of 2 representative independent biological samples. uEVs were enriched using indicated methodologies, in duplicate. uEV-enriched preparations were analysed by Western blot using antibodies against indicated proteins. Molecular weights are shown.
Mentions: Next, we evaluated several EV protein markers including CD9, CD10, CD63, TSG101, CD10, AIP1/Alix, AQP2 and FLT1. In Fig. 2, we show the WB analysis of these proteins in 2 independent biological samples (each of them in duplicated) isolated by using the 5 different methods. We found that the different methods yielded different amounts of these EV protein markers. We also noted that the electrophoretic protein mobilities obtained using INV methodology differed slightly from the protein mobilities observed for the other methods (Fig. 2). In addition, we observed clear differences between individuals, in accord with the interindividual variability shown in several studies of EVs in human biofluids. For instance, sample M3 had weaker AIP1 and CD9 bands but stronger CD10, AQP2 and CD26 bands than sample F3. To obtain quantitative data, densitometry and background subtraction for the Western blotting bands of the 10 independent samples were performed (see values in Supplementary Table III). The absolute values were transformed into relative values by referring to the highest value obtained for each protein and for each sample (Fig. 3a). For instance, NOR methodology gives a value of 100% for AIP1 in the samples shown in Fig. 2. For CD63, the 100% value was given to the levels obtained using CEN methodology. Figure 3a shows the average of the quantitative results for all samples. It was clear that NOR method resulted in a mixture enriched in AIP1 and CD26 but with lower levels of AQP2 and CD63. CEN sediments vesicles enriched in CD63, CD9 and AQP2, with lower levels of vesicles containing AIP1 (Fig. 3a). Both methods (NOR and CEN) isolated a mixture containing CD10 and FLT1, with similar efficiency. LEC-based method seemed to be very specific for CD9-positive vesicles and had the lowest efficiency for the isolation of AIP1 and CD26-containing vesicles (Fig. 3a). In contrast, INV-based methodology had high efficiency for obtaining CD26 and CD63 proteins (Fig. 3a). Remarkably, under the conditions used, EXQ method was the least efficient for most of the uEV protein markers (CD63 was detected at a relatively high level, Fig. 3a). To discard for the presence of uEVs markers as soluble proteins, we performed Western blot analysis loading NOR and CEN methodologies along with 15 µl of urine sample directly (Supplementary Fig. 3). The results showed that no detection was observed in urine sample loaded directly and uEVs protein markers were only detected when the isolation procedures were performed.

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