Comparison of methods of isolating extracellular vesicle microRNA from HepG2 cells for High-throughput sequencing
Background: Extracellular vesicles (EVs) were reported to participate in various cellular processes based on the biomolecules, particularly microRNAs. Numerous commercial EVs isolation reagents are available. However, whether these reagents are suitable for separating EVs from the culture medium sup...
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| Format: | Article |
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Frontiers Media S.A.
2022-09-01
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| Series: | Frontiers in Molecular Biosciences |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmolb.2022.976528/full |
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| author | Ying-Hui Xiong Ying-Hui Xiong Xue-Gong Fan Xue-Gong Fan Ya-Yu Chen Ya-Yu Chen Yan Huang Yan Huang Jun Quan Jun Quan Pan-Pan Yi Pan-Pan Yi Mei-Fang Xiao Ze-Bing Huang Ze-Bing Huang Xing-Wang Hu Xing-Wang Hu Xing-Wang Hu |
| author_facet | Ying-Hui Xiong Ying-Hui Xiong Xue-Gong Fan Xue-Gong Fan Ya-Yu Chen Ya-Yu Chen Yan Huang Yan Huang Jun Quan Jun Quan Pan-Pan Yi Pan-Pan Yi Mei-Fang Xiao Ze-Bing Huang Ze-Bing Huang Xing-Wang Hu Xing-Wang Hu Xing-Wang Hu |
| author_sort | Ying-Hui Xiong |
| collection | DOAJ |
| description | Background: Extracellular vesicles (EVs) were reported to participate in various cellular processes based on the biomolecules, particularly microRNAs. Numerous commercial EVs isolation reagents are available. However, whether these reagents are suitable for separating EVs from the culture medium supernatant supernatant of model cell lines, such as HepG2, and whether the isolated products are suitable for High-throughput sequencing remains unclear.Methods: We examined three commonly used EVs isolation kits: the ExoQuick-TC exosome precipitation solution (EQ), Total Exosome Isolation from cell culture medium (EI), and exoEasy Maxi Kit (EM), to isolate EVs from HepG2 cell culture medium supernatants. EVs were identified based on marker proteins, particle size measurements, and electron microscopy analysis. The total amounts of microRNA and microRNA High-throughput sequencing data quality from EVs isolated by each kit were compared.Results: The total amount of EVs’ microRNA isolated from the EI and EM groups were higher than that obtained from the EQ group (EQ/EI: p = 0.036, EI/EM: p = 0.024). High-throughput sequencing data quality evaluation showed that the EI group possessed higher quality than those in the EM group.Conclusion: For the cell culture medium from HepG2, EVs’ microRNA isolated by EI reagents might be more suitable for High-throughput sequencing applications. |
| first_indexed | 2024-04-12T18:15:21Z |
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| id | doaj.art-de7f64e0378f4ff3be71f490b06060bd |
| institution | Directory Open Access Journal |
| issn | 2296-889X |
| language | English |
| last_indexed | 2024-04-12T18:15:21Z |
| publishDate | 2022-09-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Molecular Biosciences |
| spelling | doaj.art-de7f64e0378f4ff3be71f490b06060bd2022-12-22T03:21:39ZengFrontiers Media S.A.Frontiers in Molecular Biosciences2296-889X2022-09-01910.3389/fmolb.2022.976528976528Comparison of methods of isolating extracellular vesicle microRNA from HepG2 cells for High-throughput sequencingYing-Hui Xiong0Ying-Hui Xiong1Xue-Gong Fan2Xue-Gong Fan3Ya-Yu Chen4Ya-Yu Chen5Yan Huang6Yan Huang7Jun Quan8Jun Quan9Pan-Pan Yi10Pan-Pan Yi11Mei-Fang Xiao12Ze-Bing Huang13Ze-Bing Huang14Xing-Wang Hu15Xing-Wang Hu16Xing-Wang Hu17Department of Infectious Diseases, Xiangya Hospital, Central South University, Changsha, ChinaHunan Key Laboratory of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, ChinaDepartment of Infectious Diseases, Xiangya Hospital, Central South University, Changsha, ChinaHunan Key Laboratory of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, ChinaDepartment of Infectious Diseases, Xiangya Hospital, Central South University, Changsha, ChinaHunan Key Laboratory of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, ChinaDepartment of Infectious Diseases, Xiangya Hospital, Central South University, Changsha, ChinaHunan Key Laboratory of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, ChinaDepartment of Infectious Diseases, Xiangya Hospital, Central South University, Changsha, ChinaHunan Key Laboratory of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, ChinaDepartment of Infectious Diseases, Xiangya Hospital, Central South University, Changsha, ChinaHunan Key Laboratory of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, ChinaDepartment of Health Management Center, Xiangya Hospital, Central South University, Changsha, Hunan, ChinaDepartment of Infectious Diseases, Xiangya Hospital, Central South University, Changsha, ChinaHunan Key Laboratory of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, ChinaDepartment of Infectious Diseases, Xiangya Hospital, Central South University, Changsha, ChinaHunan Key Laboratory of Viral Hepatitis, Xiangya Hospital, Central South University, Changsha, ChinaNational Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Hunan, ChinaBackground: Extracellular vesicles (EVs) were reported to participate in various cellular processes based on the biomolecules, particularly microRNAs. Numerous commercial EVs isolation reagents are available. However, whether these reagents are suitable for separating EVs from the culture medium supernatant supernatant of model cell lines, such as HepG2, and whether the isolated products are suitable for High-throughput sequencing remains unclear.Methods: We examined three commonly used EVs isolation kits: the ExoQuick-TC exosome precipitation solution (EQ), Total Exosome Isolation from cell culture medium (EI), and exoEasy Maxi Kit (EM), to isolate EVs from HepG2 cell culture medium supernatants. EVs were identified based on marker proteins, particle size measurements, and electron microscopy analysis. The total amounts of microRNA and microRNA High-throughput sequencing data quality from EVs isolated by each kit were compared.Results: The total amount of EVs’ microRNA isolated from the EI and EM groups were higher than that obtained from the EQ group (EQ/EI: p = 0.036, EI/EM: p = 0.024). High-throughput sequencing data quality evaluation showed that the EI group possessed higher quality than those in the EM group.Conclusion: For the cell culture medium from HepG2, EVs’ microRNA isolated by EI reagents might be more suitable for High-throughput sequencing applications.https://www.frontiersin.org/articles/10.3389/fmolb.2022.976528/fullextracellular vesicles isolationmicroRNAHigh-throughput sequencingHepG2 cellscell culture medium |
| spellingShingle | Ying-Hui Xiong Ying-Hui Xiong Xue-Gong Fan Xue-Gong Fan Ya-Yu Chen Ya-Yu Chen Yan Huang Yan Huang Jun Quan Jun Quan Pan-Pan Yi Pan-Pan Yi Mei-Fang Xiao Ze-Bing Huang Ze-Bing Huang Xing-Wang Hu Xing-Wang Hu Xing-Wang Hu Comparison of methods of isolating extracellular vesicle microRNA from HepG2 cells for High-throughput sequencing Frontiers in Molecular Biosciences extracellular vesicles isolation microRNA High-throughput sequencing HepG2 cells cell culture medium |
| title | Comparison of methods of isolating extracellular vesicle microRNA from HepG2 cells for High-throughput sequencing |
| title_full | Comparison of methods of isolating extracellular vesicle microRNA from HepG2 cells for High-throughput sequencing |
| title_fullStr | Comparison of methods of isolating extracellular vesicle microRNA from HepG2 cells for High-throughput sequencing |
| title_full_unstemmed | Comparison of methods of isolating extracellular vesicle microRNA from HepG2 cells for High-throughput sequencing |
| title_short | Comparison of methods of isolating extracellular vesicle microRNA from HepG2 cells for High-throughput sequencing |
| title_sort | comparison of methods of isolating extracellular vesicle microrna from hepg2 cells for high throughput sequencing |
| topic | extracellular vesicles isolation microRNA High-throughput sequencing HepG2 cells cell culture medium |
| url | https://www.frontiersin.org/articles/10.3389/fmolb.2022.976528/full |
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