Total exosome rna and protein isolation kit

CDCs (yCDC, n = 2; aCDC, n = 2) and c-kit^pos CPCs (c-kit^pos yCPC, n = 3; c-kit^pos aCPC, n = 3) were cultured for 48 hours in serum-free medium, before collection conditioned medium. Exosomes were isolated using the ultracentrifugation method. Briefly, cells were pelleted by low speed centrifugation (15 min. at 500 g). The supernatant, containing the exosomes and cell debris, were then centrifuged for 30 min. at 10,000 g to pellet the cell debris. The residual supernatant was then finally ultracentrifuged in 2 different steps (3 hours at 140,000 g, 70 min. at 140,000 g) to obtain the exosome fraction. The purified exosome pellet was dissolved in PBS for further analysis. All centrifugation and ultracentrifugation steps were performed at 4 °C. The nanoparticle analysis technology (Nanosight) was used to measure particle size and concentration. Six technical replicates per exosome sample were performed. The extraction of total RNA of the exosomes was performed using the total exosome RNA and protein isolation kit (Life technologies). The total exosome RNA yield and quality was measured by a spectrophotometer (NanoDrop2000, Thermo scientific). Normalized expression levels of miR-22-3p, miR-132-3p, miR-146a-3p and miR-210-3p were calculated using miR-16 as a housekeeping gene.

Total EVs RNA was extracted with a Total Exosome RNA and Protein Isolation Kit (Life Technology, USA). EVs miRNA expression was normalized by cel-miR-39 as a spike-in control as recommended by the manufacturer. Total cellular RNA was extracted with the miRNeasy Mini Kit (QIAGEN, Germany). U6 was employed as the internal control for cellular miRNA. For qPCR, the Mir-X™ miRNA First Strand Synthesis kit (Takara, Japan), PrimeScript™ RT Master Mix kit (Takara), and SYBR® Premix Ex Taq™ II (Takara) were used. The miRNA qPCR primer sets were purchased from RiboBio and mRNA qPCR primers were synthesized by Tsingke (Beijing, China). The qPCR primers used were listed in Supplementary Table S1.

Total RNA including miRNA from PTr2 and PAOEC cells was extracted using Total RNA extraction kit (Norgen BioTek Corp, Thorold, ON, Canada). Total RNA within EVs was isolated using Total exosome RNA and protein isolation kit (#4478545; Life Technologies Inc. Burlington, ON, Canada) according to manufacturer’s instructions. The concentration and purity of isolated total RNA was assessed using a Nanodrop 2000 C UV-Vis Spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and stored at −80 °C until further use.

Isolation of extracellular vesicles from CSF was performed with the Total Exosome Isolation Kit according to the manufacturer’s instruction (Life Technologies). Briefly, 200 μl (for RNA isolation) or 400 μl (for western blotting) of CSF were thawed on ice and centrifuged at 10,000 g for 30 min at 4°C. One volume of exosome isolation reagent was added to the supernatant and mixed by vortexing before incubating for 1 h at 4°C. Samples were centrifuged at 10,000 g for 1 h at 4°C. Supernatant was aspirated. The complete volume of supernatant was used for RNA isolation. The pellet containing extracellular vesicles was re-suspended in either 200 μl PBS (for RNA isolation; Total Exosome RNA and Protein Isolation Kit, Life Technologies) or 20 μl ice-cold Exosome Resuspension Buffer (for western blotting).