Cleanup kit

Oligonucleotides of 13 and 16 residues (sequences shown in Table 1) were purchased from The Midland Certified Reagent Company Inc. DNA samples for EMSA experiments were labeled at 5΄ termini with ³²P (25 (link)). Unincorporated [γ- ³²P]ATP was removed by buffer exchange using Sephadex spin columns equilibrated with 10 mM Tris (pH 8.0 at 21°C). DNA duplexes were prepared by annealing purified 5΄-labeled oligonucleotides with slight excess of the complementary unlabeled strands. Single-stranded DNA concentrations were measured spectrophotometrically using extinction coefficients provided by the manufacturer. Negatively-supercoiled pUC19 plasmid DNA, was obtained from New England Biolabs. This preparation was relaxed with E. coli topoisomerase I, and individual topoisomers were resolved by electrophoresis in 1.4% agarose gels (24 (link)). Linking difference values (ΔLk) were assigned by band counting (26 (link)), using the relaxed circular form as a reference with ΔLk = 0. Isolated topoisomer DNAs were purified using a polymerase chain reaction Clean-up kit from Qiagen, followed by dialysis against buffer containing 10 mM Tris (pH 8.0 at 20°C), 1 mM ethylenediaminetetraacetic acid. Plasmid DNA concentrations were measured spectrophotometrically, using ε₂₆₀ = 1.31 × 10⁴ M⁻¹cm⁻¹ (per base-pair).

Differential centrifugation was used to isolate EVs from 38.5 mL of the voided urine samples and 500 µL of serum samples. Cells were initially removed from the urine samples by centrifugation at 2,000 ×g for 30 min, following which the supernatant was centrifuged at 17,000 ×g for 30 min to remove debris and salts. After further ultracentrifugation of the supernatant at 130,000 ×g for 90 min and washing with PBS, the pellets underwent additional ultracentrifugation at 130,000 ×g for 90 min. The final obtained pellet was resuspended in 250 µL PBS and then stored at −80 °C for subsequent use. Patients’ sera were centrifuged at 300 ×g for 10 min to remove remaining cells and cell debris. After further ultracentrifugation of the supernatant at 150,000 ×g for 70 min and washing with PBS, the pellets underwent additional ultracentrifugation at 150,000 ×g for 70 min. The final obtained pellet was resuspended in 100 µL PBS and then stored at −80 °C for subsequent use. MiRNA and mRNA were isolated with a miRNeasy Mini Kit (Qiagen, Venlo, Netherlands), Clean-up Kit (Qiagen), and RNA MS2 (Roche Diagnostics, Mannheim, Germany) according to the manufacturers’ protocols.