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Hire is the method:

The goal of this study, therefore, was to identify the best isolation methods for both proteomic analysis and RNA profiling of urinary exosomes as a first step for biomarker discovery. We followed six different protocols, designated P1–P6, for the isolation of urinary exosomes, and evaluated the quantity and purity of exosomal protein and RNA using each method (Table 1). Three of the tested methods (P1–P3) were based on ultracentrifugation. P1 was the reference or standard differential centrifugation method,8 whereas P2 and P3 included an extra step to increase the purity of the isolated urinary exosomes. Specifically, P2 incorporated an additional ultracentrifugation step with a 30% sucrose cushion and P3 passed preprocessed urine through a 0.22-μm filter just before ultracentrifugation. We also tested three other methods that are faster than the ultracentrifugation-based ones: P4 was based on the use of Vivaspin 20 nanomembrane ultrafiltration concentrator (Sartorius; Bohemia, NY),12 whereas P5 and P6 utilized the exosome precipitation reagent ExoQuick-TC (System Biosciences; Mountain View, CA), which is specific for the isolation of exosomes from both urine and cell cultures. To our knowledge, this is the first report that evaluates differences in yield and quality of exosomal protein, miRNA, and high-molecular weight (HMW) RNA obtained using multiple methods of exosome isolation from urine, including two different protocols based on the ExoQuick-TC reagent.

Table 1. Exosome isolation protocols

Protocol name

Method

P1

Ultracentrifugation

P2

Ultracentrifugation+30% sucrose cushion

P3

Ultracentrifugation+0.22 μm filtration

P4

Nanomembrane ultrafiltration concentrator (Vivaspin 20; Sartorious)

P5

Standard exosome precipitation (ExoQuick-TC; System Biosciences)

P6

Modified exosome precipitation (ExoQuick-TC; System Biosciences)

RESULTS

Reduction of Tamm–Horsfall protein (THP) to increase the yield of urinary exosomes

We first sought to optimize the overall yield of exosomes from urine samples. Urine is rich in large amounts of a polymeric network formed by the Tamm–Horsfall protein (THP), which traps exosomes during the initial low-speed centrifugation (17,000 g) of the urine preprocessing stage.16 To account for this effect and potentially increase exosome yield, we reduced THP using DL-dithiothreitol during the preprocessing of urine for P1–P4 and P6 (Figure 1) and the isolation of exosomes (P1–3, Figure 2), as described in the Materials and Methods section. As shown in Figure 3a and b, a higher amount of THP was present in the 17,000 g pellet obtained from P5, compared with the DL-dithiothreitol-treated protocols. As shown in Figure 3c, a higher amount of THP is observed in the 17,000 g pellet using P5 compared with P6, and no THP was present in the supernatant obtained after the last centrifugation using both protocols.

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Figure 1. Urine preprocessing. Urine was centrifuged at 17,000 g (P1, P2, P3, P4, and P6) or at 3000 g (P5) to remove urinary sediment including whole cells, large membrane fragments, and other debris. To reduce the entrapment of exosomes by Tamm–Horsfall protein, we dissolved pellets in 500 μl isolation solution (I.S.; 250 mmol/l sucrose, 10 mmol/l triethanolamine, pH 7.6), added 100 mg of DL-dithiothreitol (DTT), and incubated them for 10 min at 37 °C, and then centrifuged again at 17,000 g for 10 min at 37 °C. The supernatant (second supernatant (SN2)) from protocols 1, 2, 3, 4, and 6 was combined with SN1 (first supernatant) and processed as summarized in Figure 2. P1, P2, and P3 are exosome isolation protocols based on ultracentrifugation with either an extra purification step with 30% sucrose cushion (P2) or a 0.22-μm filtration following the urine preprocessing step (P3). P4 uses the Vivaspin 20 nanomembrane filtration concentrator (Sartorius), and P5 (standard method) and P6 (modified method) are based on the exosome precipitation reagent ExoQuick-TC (System Biosciences). PIC, protease inhibitor cocktail (Sigma); SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

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Figure 2. Isolation of urinary exosomes. After urine was preprocessed as described in Figure 1, six different protocols were used to isolate exosomes, three based on ultracentrifugation at 165,000 g (P1, P2, and P3), one on the use of the Vivaspin 20 nanomembrane filtration concentrator (Sartorius; P4), and two that use the exosome precipitation reagent ExoQuick-TC (System Biosciences); P5 is the standard method recommended by the manufacturer, whereas P6 is our modification of that method. DTT, DL-dithiothreitol; I.S., isolation solution (250 mMsucrose, 10 mM triethanolamine, pH 7.6); PBS, phosphate-buffered saline (0.14 M NaCl, 0.0027 M KCl, 0.010 M PO43−, pH 7.4); SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SN, supernatant. Nanomembrane filtration concentrator Vivaspin 20, 100 kDa MWCO (Sartorious; Bohemia, NY).

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Figure 3. Protein electrophoretic patterns of different urinary fractions. The urinary sediment obtained after the last 17,000 g spin (protocols P1–P4, and P6) or the first 3000 g spin (P5), with or without DL-dithiothreitol (DTT) treatment as indicated in the figure, was resuspended in 30 μl of reduced sample mix and loaded on a NuPAGE Novex 4–12% Bis-Tris gel and stained with silver as described in the Materials and Methods section. (a, b) Analysis of two different spot urine samples provided by two healthy subjects. One urine sample (a) is more concentrated than the other one (b), and therefore a lower number of protein bands are visible in the silver-stained gel. (c) Comparison of exosome pellet (E), 17,000 g or 3000 g pellet (P; urinary sediment), and the last supernatant (SN3, Figure 2) obtained using exosome isolation protocols P5 and P6, which are both based on the exosome precipitation reagent ExoQuick-TC. The large 85–100-kDa band corresponds to the Tamm–Horsfall protein (THP) monomer, which consists of approximately 70% protein and 30% carbohydrate by weight.27 M, protein molecular marker; U, unprocessed urine.

Quantification of urinary exosome particles

We next quantified the number of urinary exosome particles using CD9 enzyme-linked immunosorbent assay (ELISA). As shown in Figure 4a, the highest number of exosome particles per ml of urine (∼2.7 × 109) was obtained using P6, followed by P1 and P4. We also found that the yield of exosome particles per ml of urine was more than five times higher using the modified ExoQuick-TC (P6) compared with the standard ExoQuick-TC (P5). The lowest yield of exosome particles was obtained using P2.

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Figure 4. Quantification of the number of exosomal particles and total exosomal protein. (a) Estimation of exosome particle number per ml of urine by CD9 enzyme-linked immunosorbent assay. Bars are mean±s.d. of four different spot urine samples provided by four healthy subjects. (b) Quantification of total exosomal proteins per ml of urine by absorbance measurement at 280 nm. **P

Hello Sneha

Our group use ExomiR extraction kit. You could try with this.

Best regards

Hello Mr.Oscar,

I have performed the exosome isolation using the ultracentrifugation process and also validated it's size using the nanosight. I just want to know a good way of quantifying the miRNA concentration other than fragment analyzer. Any inputs or suggestions would be appreciated.

Thanks,