A Method Of Serum Exosome Isolation And Its Application For Breast Cancer Microrna Detection

Objectives: Exosomes are small membrane bound vesicles(30-150 nm)containing various functional proteins,m RNAs and micro RNAs(mi RNAs)that could be used for diagnostic and therapeutic purposes.Exosomes are secreted by almost all cell types in culture and in vivo at both normal and diseased states.Currently,the standard method for serum exosome isolation is differential ultracentrifugation,while the search for alternative,less time-consuming and labor extensive exosome isolation method for use in clinical settings is still ongoing.Here,we aim to set up a simple and low-cost method to isolate exosomes from serum and detect micro RNAs enclosed in these circulating exosomes.Methods: Exosomes were isolated from blood serum of healthy individuals or breast cancer patients by ultracentrifugation,Exo Quick Precipitation method and our method.The particle size of exosomes was measured by Nanosight NS90(Marvern)and TEM(FEI,Talos F200X).The level of exosome protein marker CD9 was determined by Western blot.The expression levels of mi R-21 in serum and circulating exosomes of breast cancer patients and healthy individuals were determined by real time PCR.Results: Regarding particle size and concentration,the performance of our method was better than ultracentrifugation,and equivalent to other commercial products.Using our method and reagent,we tested the mi R-21 expression level from serum and circulating exosomes of breast cancer patients and healthy persons.Compared with benign tumor,mi R-21 was up-regulated by 2.68 fold(P = 0.024)in circulating exosomes of malignant breast cancer patients.However,the mi R-21 expression level was almost equal in serum samples between the benign and malignant breast tumor patients.Conclusions: Our method is easier to operate than ultracentrifugation,quicker and lower in cost than other commercial products.We also provide a new method for reference to researchers in the exosome field,by which exosome isolation from serum samples and detection of mi RNAs in circulating exosomes can be achieved.The comparison of mi R-21 levels in serum and isolated exosomes from benign and malignant breast tumors also indicated that measurement of mi RNAs in exosomes could be a more specific method than that in serum.