Since exosomes are derived from multivesicular endosomes or MVB-containing lipid bilayer membrane structures trapped in the cytoplasmic membrane, they are present in the extracellular matrix. In order to obtain exosomes with high purity for Exosome RNA sequencing, it is necessary to ensure effective removal of all cellular debris and other unwanted membrane structures.
Blood-based samples contain serum and plasma. For blood samples, hemolysis is a major problem that cannot be ignored. The occurrence of hemolysis means the rupture of red blood cells. Blood samples with hemolysis are not recommended for further use, so it is important to handle blood samples promptly after collection and avoid violent shaking.
Differences between serum and plasma samples
Serum: The liquid released by the clotting of blood after the clotting of isolated blood. There is no fibrinogen or clotting factors, but platelets secrete large amounts of exosomes during the clotting process, so serum samples are preferentially recommended for the study of platelet-related diseases.
A brief procedure for the pretreatment of blood samples
Plasma: The fluid obtained from whole blood leaving the blood vessels after anticoagulation and precipitation by centrifugation, which does not contain cellular components. However, the biggest disadvantage of samples of plasma is the interference of anticoagulants.
A brief procedure for the pretreatment of blood samples
Briefly, the difference between serum and plasma is the lack of fibrinogen and coagulation factors in the former with the added contamination of exosomes released from a large number of platelets.
These samples mainly include urine, cerebrospinal fluid, pleural effusion, alveolar lavage, thoracic lavage, saliva, amniotic fluid, breast milk, etc. Samples of body fluids other than blood are prone to bacterial contamination or cellular RNA contamination, and require strict centrifugation steps to remove the supernatant, preferably by filtration and decontamination. Except for urine, the volume of body fluids that can be obtained is generally relatively small. Therefore, the amount of RNA obtained by extraction is also relatively small.
Taking urine as an example, we would like to share with your pretreatment considerations:
Cell supernatant is a very well-studied sample that yields a high amount of RNA for subsequent experiments due to the relatively low difficulty in obtaining it. In our project experience, the total RNA obtained from cell supernatant-derived exosome extraction can reach tens of micrograms or even more, but in some cases only pico-scale amounts of total RNA are obtained. This is related to the volume of the cell supernatant, the cell culture method, and the ability of the cells themselves to secrete exosomes.
Since the cell culture process is prone to mycoplasma contamination, it is recommended to treat cells with mycoplasma removal reagents and test for common mycoplasmas before sending samples.
Recommended sample volume: 40 mL or more
Caution
For exosome RNA sequencing, the number of samples to be tested should be determined based on various factors, such as the research purpose, sample source, and budget. In general, at least three pairs of samples are recommended, but the specific number should be determined based on the actual situation.
If the extracellular vesicle (EV) RNA sequencing is carried out on cell culture supernatant, fewer samples may be required because the background of the cell model is relatively uniform. However, for clinical samples, more samples are required, and at least five pairs or even more are recommended.
For studies requiring higher precision and statistical significance, such as biomarker research, a larger number of samples are needed. When funding is limited, a strategy of using pilot experiments and small sample validation can be considered.
In summary, the selection of the number of samples needs to fully consider various factors such as the purpose of the experiment, budget, and technical requirements. At the same time, in experimental design, attention should be paid to details such as sample source, sample preservation, and processing to ensure the reliability and reproducibility of the experimental results.