A method for isolating exosomes from animal plasma and performing purity testing
A technology of purity detection and exosomes, which is applied in the field of cell biology to achieve high biological activity
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Embodiment 1
[0047] A 2.7Kg rabbit was collected 10ml of blood by way of jugular vein blood collection, and the anticoagulant was EDTA. The anticoagulated blood was transferred into a 2ml centrifuge tube and centrifuged at 3000rpm for 30min to separate the plasma. The separated plasma was used for exosome extraction.
[0048] Methods for isolating exosomes from rabbit plasma, such as figure 1 shown, including the following steps:
[0049] (1) Dilute 2ml of plasma with PBS solution at a ratio of 1:1;
[0050] (2) Centrifuge at 2000g for 30min at 4°C to remove cellular components in the plasma, and retain the supernatant after centrifugation;
[0051] (3) Collect the filtrate into a centrifuge tube again, and centrifuge at 12,000 g for 30 minutes at 4°C to remove cell debris. If the plasma needs to be frozen, store it at -80°C after this step;
[0052] (4) Collect the supernatant into a centrifuge tube, filter the supernatant with a 0.22 μm filter membrane, so that microcapsules smaller ...
Embodiment 2
[0061] The exosomes isolated in Example 1 were subjected to total RNA extraction, miRNA quantification and purity detection, and the miRNA extracted in Example 1 was subjected to poly(A) reverse transcription and quantification.
[0062] The present invention utilizes 6 highly expressed miRNAs identified in human plasma for miRNA quantitative detection, namely miR-93a-3p, miR-451a-5p, miR-21-5p, miR-16-5P, miR-181 -5p, miR-92a; two additional intracellular genes were used: U6 and 5S. U6 is a small nuclear molecule RNA, which mainly exists in the nucleus. 5S rRNA is a component of the large ribosomal subunit. Both prokaryotes and eukaryotes have 5S rRNA, and their structures are similar. Both types of genes are not present in exosomes and thus serve as negative controls. The primer sequences of the 12 genes are shown in the list. Specific steps are as follows:
[0063] Poly(A) tailing reaction reverse transcribes miRNA into cDNA. Specific steps are as follows:
[0064] (1...
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