Complete seramir exosome rna amplification kit

The sEVs from the cultivation media were isolated using the Complete SeraMir Exosome RNA Amplification kit (System Biosciences, Mountain View, CA, USA) containing Exoquick-TC (System Biosciences, Mountain View, CA, USA), using the protocol recommended by the supplier. Briefly, CM was centrifuged at 3000× g for 15 min to remove cells and cell debris. Then, 5 mL of supernatant was mixed with 1 mL of Exoquick solution, mixed, and then incubated overnight at 4 °C. Subsequently, the tubes were centrifuged at 16,000× g for 2 min, and the sEVs pellet was reconstituted in 350 μL of lysis buffer. After vortexing, followed by incubation for 5 min at room temperature to allow for complete lysis, 200 μL of 100% ethanol was added, and 600 μL of the mixture was filtered through the spin column at 16,000× g for 1 min. Two wash steps were then performed with 400 μL of wash buffer, followed by centrifugation at 16,000× g for 1 min, which was then followed by the elution of total RNA into 30 μL of elution buffer. The quality of the eluted exoRNA was then evaluated using the Agilent RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA, USA).

Ad-sEV total RNA was extracted using mirVana microRNA Isolation kits (Life Technologies, Carlsbad, CA, USA). Total RNA was amplified with the Complete Seramir Exosome RNA Amplification Kit (System Biosciences, Mountainview, CA, USA). RNA quality was assessed on a subset of these samples using the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) and Nanodrop 2000 (ThermoFisher Scientific, Waltham, MA, USA). RNA concentration was measured using a Qubit RNA Broad Range Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).

Five microliters of the eluate was then used for reverse transcription using the Complete SeraMir Exosome RNA Amplification kit (System Biosciences, Mountain View, CA, USA). For the poly A reaction, exoRNA eluted from the spin column (5 µL), 5× polyA buffer (2 µL), 25 mM MnCl₂ (1 µL), 5 mM ATP (1.5 µL), and polyA polymerase (0.5 µL) were incubated for 30 min in 37 °C. Then, 0.5 µL of SeraMir Adaptor Oligo was added, and the mix was incubated for 5 min at 60 °C. After subsequent incubation at room temperature for 2 min, the RT reaction was performed in a 20 µL volume and consisted of polyA exoRNA from the previous step (10 µL), 5× RT master mix (4 µL), 5’ SeraMir Switch Oligo (1 µL), reverse transcriptase (1 µL), and RNA-se-free water (4 µL). Incubation in a BioRad T100 thermal cycler (Bio-Rad, Hercules, CA, USA) at 42 °C for 30 min was followed by 95 °C for 10 min to terminate the reaction.