Total exosomal protein and rna isolation kit

Exosomal RNA was isolated using the total exosome RNA and protein isolation kit (Life Technologies) as per the manufacturer’s instructions. The total RNA concentration was measured using the NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Isolated exosomes were re-suspended in exosome resuspension buffer as per the total exosomal protein and RNA isolation kit (Life Technologies). The total protein concentration was determined using the RC DC Protein Assay as per manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA, USA).

Exosomes were isolated using the exosome miRCURY isolation kit (Qiagen, USA) according to the manufacturer’s instruction with modifications. Briefly, plasma was centrifuged at 300 g for 10 min the supernatant was then centrifuged at 2000 g for 30 min to remove dead cells. Thereafter the recovered supernatant was centrifuged at 12,000 g for 45 min to remove cell debris. To ensure removal of all unwanted cells and debris the supernatant was further purified using a 0.22 µm spin column (Corning, USA). Thrombin was then added to the filtrate for de-fibrination. This was followed by centrifugation at 10 000 g for 5 min. Precipitation buffer was thereafter added to the recovered supernatant and incubated at 4 ºC for 4hrs. Post incubation the exosomes were recovered by centrifugation at 500 g for 5min at 20 ºC. The exosomal pellet was resuspended in exosome resuspension buffer as prescribed by the total exosomal Protein and RNA isolation kit (Life Technologies).