Exoeasy maxi kit

A circRNA array pattern analysis of exosomes derived from normoxia or hypoxia pericytes was performed by OE Biotech Company (Shanghai, China). Briefly, exosomal RNAs were isolated using a QIAGEN exoEasy Maxi Kit (QIAGEN GmbH, Germany) and then digested with RNase R (Epicentre Biotechnologie, Inc., Madison, WI, USA) to remove linear RNAs. The obtained enriched circRNAs were labeled using reagents in an Arraystar Super RNA Labeling Kit (Arraystar, Rockville, MD, USA) and then hybridized onto an Arraystar Human circRNAs chip (Arraystar). For microarray analysis, the hybridized arrays were scanned with an Agilent G2565CA Microarray Scanner System (Agilent Technologies, Santa Clara, CA, USA). Agilent Feature Extraction software was used to perform quantile normalization and data preprocessing. Differentially expressed circRNAs were defined as those with a p value < 0.05 and a > 1.5 fold-change in expression as determined from a comparison between normoxia and hypoxia exosome groups. The results were analyzed by hierarchical clustering.

Purifications of exosomes from serum-free cell culture supernatants were carried out via exoEasy Maxi Kit (Qiagen^®). In contrast to conventional ultracentrifugation method, the membrane affinity spin columns method allows for rapid isolation time with reported improved in quantity and purity of isolated exosomes. Briefly, the harvested cell culture supernatant was filtered from any cellular fragments and cell debris through 15 min centrifugation at 3000× g and at 4 °C. Then, they were mixed with buffer XBP in a 1:1 ratio and were centrifuged at 500× g for 1 min via the exoEasy™ spin column, prior to discarding the flow-through. Ten mL of buffer XWP was then added, and the spin columns were centrifuged at 5000× g for 5 min to remove any residual buffer, prior to discarding the flow-through. After transferring the spin column to a fresh collection tube, 500 µL of buffer XE was added directly onto the membrane and the spin column was incubated for 1 min, before they were centrifuged at 500× g for 5 min to collect the elute.

Exosomes in SCC9 and CAL27 cells were isolated using the exoEasy Maxi Kit (Qiagen, Germany). First, cell culture was centrifuged for 20 min at 3000 × g to remove cells and cellular debris. Next, the supernatant was filtered to remove particles over 0.8 μm followed by mixing with Buffer XBP. Thereafter, the mixture was bound to exoEasy membrane affinity spin column. Extracted exosomes were rinsed by Buffer XWP and then washed by Buffer XE. Afterwards, exoRNeasy Midi and Maxi Kit(Qiagen) was applied for extracting RNAs.