Extraction of exosome by gel electrophoresis microfluidic chip and determination of miRNA-21 in exosome of human plasma.

Dan Luo; Fengying Ran; Lun WU; Juan Zhang; Fangling Ren; Jingjian Liu; Binqiang Zhang; Qinhua Chen

Return

Extraction of exosome by gel electrophoresis microfluidic chip and determination of miRNA-21 in exosome of human plasma.

Author: Dan LUO ¹ ; Fengying RAN ¹ ; Lun WU ¹ ; Juan ZHANG ² ; Fangling REN ¹ ; Jingjian LIU ¹ ; Binqiang ZHANG ¹ ; Qinhua CHEN ¹
Author Information

1. Department of Medical Experimental Center, Affiliated Dongfeng Hospital of Hubei University of Medicine, Shiyan 442008, Hubei, China.
2. College of Chemistry and Environmental Engineering, Wuhan Institute of Technology, Wuhan 430205, Hubei, China.
Publication Type:Journal Article
Keywords: cancer; exosomes; gel electrophoresis; microfluidic chip; nanoporous membrane
MeSH: Exosomes; Humans; MicroRNAs/genetics*; Microfluidics; Plasma; Ultracentrifugation
From: Chinese Journal of Biotechnology 2021;37(2):663-672
CountryChina
Language:Chinese
Abstract: We developed a high-efficiency microfluidic chip for extracting exosomes from human plasma. We collected peripheral blood from normal human, designed and fabricated a microfluidic chip based on nanoporous membrane and agarose gel electrophoresis to isolate exosomes. The extracted exosomes were characterized by transmission electron microscopy, nanosight and Western blotting, the morphology, concentration and particle size of exosomes were identified and analyzed. Meanwhile, we used ultracentrifugation and microfluidic chip to isolate exosomes separately. The particle size and concentration of the exosomes extracted by two methods were compared and analyzed, and their respective extraction efficiency was discussed. Finally, the expression level of miRNA-21 in exosomes was analyzed by RT-PCR. The microfluidic chip isolated (in 1 hour) high-purity exosomes with size ranging from 30-200 nm directly from human plasma, allowing downstream exosomal miRNA analysis. By comparing with ultracentrifugation, the isolation yield of microfluidic chip was 3.80 times higher than ultracentrifugation when the volume of plasma sample less than 100 μL. The optimized parameters for exosome isolation by gel electrophoresis microfluidic chip were: voltage: 100 V; concentration of agarose gel: 1.0%; flow rate of injection pump: 0.1 mL/h. The gel electrophoresis microfluidic chips could rapidly and efficiently isolate the exosomes, showing great potential in the research of exosomes and cancer biomarkers.