Plasma serum exosome purification kit

After propensity matching, a total of 6 sample groups were selected (AIS patients = 6, healthy individuals = 6) for exosome-derived miRNA sequencing analysis. Following sample thawing, exosomes were purified using the Plasma/Serum Exosome Purification Kit (Qiagen, Norgen Biotek, Canada). Exosome-derived miRNA was extracted using the Exosomal RNA Isolation Kit (Qiagen, Norgen Biotek, Canada) according to the manufacturer's protocol. The extracted exosome-derived miRNA was then amplified. TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, USA) were employed for library preparation. Sequencing was carried out using the Illumina Hiseq2000/2500 platform with a single-end sequencing read length of 1 × 50 bp.

The 4 ml plasma samples were rapidly thawed at 37°C. Exosome isolation and purification were performed using the Plasma/Serum Exosome Purification Kit (Qiagen, Norgen Biotek, Canada) following the manufacturer's instructions. Initially, the plasma was diluted by adding 12 ml of nuclease-free water, followed by the addition of 300 μl of ExoC Buffer and 400 μl of Slurry E. After being incubated at room temperature for 5 min, the mixture was centrifuged at 2,000 revolutions per minute (RPM) for 2 min to remove the supernatant. Subsequently, 400 μl of ExoR Buffer was added, and the mixture was incubated for 10 min. Afterwards, it was centrifuged using a spin column at 6,000 RPM for 1 min, resulting in purified exosomes.