Method for extracting exosome from yeast cells
A technology of yeast cells and exosomes, applied in the biological field, to achieve the effect of large market prospects and application value
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Embodiment 1
[0018] 1 material
[0019] Culture medium RPMI was purchased from Gibco Company; bovine serum (fetal bovine serum, FBS) was purchased from Sigma Company; trypsin and EDTA were purchased from Invitrogen Company.
[0020] 2 methods
[0021] 2.1 Obtaining yeast cells
[0022] Yeast cells were incubated with RPMI medium containing 10% fetal bovine serum in 5% CO 2 , 37°C in an incubator with saturated humidity for primary culture, digested with 0.25% trypsin digestion solution to pass to 3 generations, and then purified by repeated adherence and trypsin digestion alternately to remove, and finally the cell line was obtained. Wherein, the 1:4 mixture of 0.05% EDTA and 0.3% trypsin was used to incubate in a 37° C. incubator for 15-20 minutes during passage, and the pH value of the culture solution was 7.4-7.5.
[0023] 2.2 Treatment of yeast cell culture supernatant
[0024] Collect the cell culture supernatant and centrifuge at room temperature or 4°C at 3500g for 5 minutes to ...
Embodiment 2
[0027] Exosome protein identification of embodiment 2 yeast cells
[0028] Phosphate buffer was added to the exosomes of yeast cells to prepare a suspension. Take 1ml of its suspension, add 2 μL Triton-100 (polyethylene glycol octylphenyl ether), and 5 μL protease inhibitor cocktail to lyse on ice for 30 minutes. Centrifuge at 15,000 g for 60 minutes at 4°C, and the supernatant is the exosome protein lysate. Take 1.0 μg of salivary exosome protein for HPLC detection, and use q-TOF-MS / MS to identify the peptides of yeast cell exosomes. The method of the present invention obtains more peptide components.
[0029] Table 1 Comparison of the method of the present invention and the centrifugation method for extracting exosome protein
[0030] method Yeast exosomal protein SD RSD(%) The method of the invention 8.23±1.2 0.056 1.28 centrifugation method 5.84±0.9 0.081 3.55
[0031] It can be seen from Table 1 that the method of the present inventio...
Embodiment 3
[0032] Example 3 Detection of Exosomal RNA Content in Yeast Cells
[0033] RNA was extracted from exosomes obtained above using the Trizol method, and the RNA concentration and quality were detected using the Agilent 2200 nucleic acid automated electrophoresis system according to the instruction manual of the High Sensitivity RNA detection reagent, as follows:
[0034] Take 1 μL of the corresponding RNA sample, 4 μL sample buffer (5 μL in total) and mix well. The mixed samples were incubated in a PCR machine at 72°C for 3 minutes, placed on ice for 2 minutes after incubation, and briefly centrifuged to concentrate the samples at the bottom of the 8-tube tube. Put the 8 tubes into the sample tank of the Agilent220 nucleic acid automatic electrophoresis system for automatic electrophoresis. The results are shown in Table 2.
[0035] Table 2 Detection of exosomal RNA content in yeast cells
[0036] small RNA species content Proportion miRNA 3620 34％ tR...
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