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
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
Blood collection with blood collection needles and ordinary blood collection tubes (without any reagents) (moved to 4°C environment for resting within 10min)
3 to 4 hours at 4 °C, visible clot precipitation
Pipette the above yellowish serum to 15ml centrifuge tube, 4000 g, 4°C centrifugation for 10min, remove the supernatant can then 1500 g, 4°C centrifugation for 10min, to maximize the quality of serum
Centrifuge the serum as soon as possible after freezing in the -80°C refrigerator
Recommended sample volume: 3 mL or more
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
Draw whole blood with a blood collection needle and anticoagulation tube (containing EDTA) and mix gently
Centrifuge at 4000 g for 5 min at 4°C, and take the supernatant as plasma, taking care not to touch the bottom and side sediment
Freeze at -80℃ before sending the sample
Recommended sample volume: 4mL or more
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.
Body Fluids Samples Other Than Blood
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:
Use 15 mL or 50 mL centrifuge tubes to collect urine, preferably mid-phase urine
If using 15 mL or 50 mL centrifuge tubes: 1000-1500 g, centrifuge at 4℃ for 30min to remove cell debris and other impurities, take the supernatant and recommend to continue filtering with 0.45μm filter head to remove bacteria
Freeze at -80°C before sending the sample
Recommended sample volume: 40mL or more
Cell Supernatant
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.
When cells are cultured in normal serum-containing medium for a certain period of time, the density of walled cells reaches 70% -80% and the density of suspended cells reaches 60% -70%.
For walled cells, remove medium and wash 1-3 times with PBS; for suspension cells, collect cells at 300 g, 4°C, for 10 min
Add new medium without exosomes or use serum-free medium and continue to culture for 48h~72h, collect cell supernatant according to cell growth rate
Collect cell supernatant, 700 g, centrifuge at 4°C for 10 min, remove residual cells, take supernatant, 9000 g, centrifuge at 4°C for 15 min, completely remove cell debris, take supernatant (be careful to avoid inhaling cells or cell debris)
Freeze and store in -80°C refrigerator before sending the sample
Recommended sample volume: 40 mL or more
Caution
No RNA protector (e.g. Trizol, RNA later) should be added to the sample before exosome isolation;
If exosomes are separated for particle size detection or flow identification, they should be stored at 4°C and the experiment should be performed as soon as possible;
The number of exosomes enriched from different sample sources varies greatly, and therefore, the total amount of extracted RNA also varies greatly. Relatively speaking, serum and plasma exosome samples have higher exosome content.
How Many Samples Are Needed for Exosome RNA Sequencing?
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.
* For Research Use Only. Not for use in diagnostic procedures.
