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For biomarker miRNAs, what are the differences between extracting from serum, exosomes, or whole blood? Which one is better?
For most studies involving Exosome RNA isolation, we recommend using plasma. Serum is the liquid collected after blood coagulation, so it lacks fibrinogen and clotting factors, and contains many coagulation products. Fibrinogen converts to fibrin, which has clotting function. During serum collection and coagulation, platelets are stimulated and release many exosomes and other vesicles. Therefore, the number of vesicles obtained from serum is always higher than from plasma, with over 50% potentially derived from platelets. Thus, plasma is a better medium for studying exosomes in pathophysiological states. Therefore, plasma is generally chosen for experiments. However, when studying diseases related to platelets, serum should be prioritized.
During blood collection, cells and platelets should be removed by prompt centrifugation, usually within 30 minutes, avoiding long storage. Also, plasma (or serum) should be separated at room temperature. The centrifugation speed and rotor type should be consistent for all samples.
For detecting exosomal protein markers by flow cytometry, are beads necessary? Are there specific steps?
Flow cytometry can detect exosomal marker proteins. Specific steps are as follows:
(1) Process the sample and extract exosomes.
(2) Antibody incubation: Resuspend the extracted exosomes in PBS containing 2% BSA. Perform primary and secondary antibody incubations sequentially to obtain the CD63-labeled exosome detection solution.
(3) Set flow cytometer parameters: Run the flow cytometer normally and set the parameters.
(4) Detection: Resuspend the CD63-labeled exosome detection solution in PBS and load it for detection.
(5) Use flow cytometry analysis software to analyze the data and obtain quantitative detection results for exosomes.