Real-time quantitative PCR array analysis of primary colon cancer and liver metastasis derived exosomes. Mus musculus

To isolate exosomes from colon tissue, colon tissue were removed and gently disaggregated with tweezers in dissociation buffer, followed by incubation at 37°C for 1 h. The disaggregated samples were centrifuged at 1,000 g for 10 min, 2,000 g for 20 min, 4,000 g for 30 min and 10,000 g for 1 h with the supernatant being retained each time. The tissue exosomes were collected by centrifuging the samples at 100,000 g for 1.5 h at 4°C, and the pellet was suspended in ice-cold PBS. Total RNA was isolated from cells, exosomes and tissue using a miRNeasy mini kit (Qiagen). miRNA expression profiling including exosomes and their donor cells or tissues was performed using the Qiagen miScript miRNA PCR Array Mouse miRBase Profiler. To isolate exosomes from liver tissue, a 20-G catheter was inserted into the portal vein of anaesthetized mice. The perfused liver tissue, as well as colon tissue were removed and gently disaggregated with tweezers in dissociation buffer, followed by incubation at 37°C for 1 h. The disaggregated samples were centrifuged at 1,000 g for 10 min, 2,000 g for 20 min, 4,000 g for 30 min and 10,000 g for 1 h with the supernatant being retained each time. The tissue exosomes were collected by centrifuging the samples at 100,000 g for 1.5 h at 4°C, and the pellet was suspended in ice-cold PBS. Total RNA was isolated from cells, exosomes and tissue using a miRNeasy mini kit (Qiagen). miRNA expression profiling including exosomes and their donor cells or tissues was performed using the Qiagen miScript miRNA PCR Array Mouse miRBase Profiler. Overall design: qPCR miRNAs expression profiling. One million CT26 colon cancer cells were administered into intra splenic injection or colonic submucosa via endoscopic work station. On day 3, 7, and 14 mice were sacrificed and tumors were removed and miRNA expression profiling including exosomes and their donor cells or tissues was performed using the Qiagen miScript miRNA PCR Array.