A Protocol for Cancer-Related Mutation Detection on Exosomal DNA in Clinical Application
BackgroundRecently, some genomic mutations in exosomal DNA have been found to be related to disease progress and clinical outcomes of patients in several cancers. Unfortunately, the methods for exosome isolation and exosomal DNA analysis are still lack of relevant research to ensure their optimal pe...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2020-09-01
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| Series: | Frontiers in Oncology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fonc.2020.558106/full |
| _version_ | 1811337038175141888 |
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| author | Zhe-Ying Wang Rui-Xian Wang Xiao-Qing Ding Xuan Zhang Xiao-Rong Pan Jian-Hua Tong |
| author_facet | Zhe-Ying Wang Rui-Xian Wang Xiao-Qing Ding Xuan Zhang Xiao-Rong Pan Jian-Hua Tong |
| author_sort | Zhe-Ying Wang |
| collection | DOAJ |
| description | BackgroundRecently, some genomic mutations in exosomal DNA have been found to be related to disease progress and clinical outcomes of patients in several cancers. Unfortunately, the methods for exosome isolation and exosomal DNA analysis are still lack of relevant research to ensure their optimal performance and the comparability. Here we aim to establish a protocol for cancer-related mutation detection on exosomal DNA in clinical application.MethodsTaking KRAS mutation in pancreatic cancer as an example, we tested whether the types of blood samples, the potential factors in the courses of exosome isolation and exosomal DNA preparation, as well as the detail in mutation detection by droplet digital PCR (ddPCR) could influence the exosomal DNA analysis.ResultsWe found that the concentration of exosomal DNA from serum was higher than that from plasma, whereas the mutant allele fraction (MAF) of KRAS in serum-derived exosomal DNA was obviously lower. The membrane-based method for exosome isolation showed no evident difference in both exosomal DNA yield and KRAS MAF from the classical ultracentrifugation method. DNase I pretreatment on exosomes could remove the wild-type DNA outside of exosomes and increase the KRAS MAF. PBS might interfere with the effect of DNase I and should not be recommended as resuspension buffer for exosomes if the subsequent experiments would be done with exosomal DNA. Besides, the denaturation of exosomal DNA before droplet generation during ddPCR could effectively improve the total KRAS copy number and mutation-positive droplet number.ConclusionThis study provides some methodological evidences for the selection of the optimal experimental conditions in exosomal DNA analysis. We also suggest a protocol for mutation detection on exosomal DNA, which might be suitable for the clinical testing and could be helpful to the comparison of results from different laboratories. |
| first_indexed | 2024-04-13T17:48:13Z |
| format | Article |
| id | doaj.art-cbba59582e43441484bff872f8ab72f8 |
| institution | Directory Open Access Journal |
| issn | 2234-943X |
| language | English |
| last_indexed | 2024-04-13T17:48:13Z |
| publishDate | 2020-09-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Oncology |
| spelling | doaj.art-cbba59582e43441484bff872f8ab72f82022-12-22T02:36:51ZengFrontiers Media S.A.Frontiers in Oncology2234-943X2020-09-011010.3389/fonc.2020.558106558106A Protocol for Cancer-Related Mutation Detection on Exosomal DNA in Clinical ApplicationZhe-Ying WangRui-Xian WangXiao-Qing DingXuan ZhangXiao-Rong PanJian-Hua TongBackgroundRecently, some genomic mutations in exosomal DNA have been found to be related to disease progress and clinical outcomes of patients in several cancers. Unfortunately, the methods for exosome isolation and exosomal DNA analysis are still lack of relevant research to ensure their optimal performance and the comparability. Here we aim to establish a protocol for cancer-related mutation detection on exosomal DNA in clinical application.MethodsTaking KRAS mutation in pancreatic cancer as an example, we tested whether the types of blood samples, the potential factors in the courses of exosome isolation and exosomal DNA preparation, as well as the detail in mutation detection by droplet digital PCR (ddPCR) could influence the exosomal DNA analysis.ResultsWe found that the concentration of exosomal DNA from serum was higher than that from plasma, whereas the mutant allele fraction (MAF) of KRAS in serum-derived exosomal DNA was obviously lower. The membrane-based method for exosome isolation showed no evident difference in both exosomal DNA yield and KRAS MAF from the classical ultracentrifugation method. DNase I pretreatment on exosomes could remove the wild-type DNA outside of exosomes and increase the KRAS MAF. PBS might interfere with the effect of DNase I and should not be recommended as resuspension buffer for exosomes if the subsequent experiments would be done with exosomal DNA. Besides, the denaturation of exosomal DNA before droplet generation during ddPCR could effectively improve the total KRAS copy number and mutation-positive droplet number.ConclusionThis study provides some methodological evidences for the selection of the optimal experimental conditions in exosomal DNA analysis. We also suggest a protocol for mutation detection on exosomal DNA, which might be suitable for the clinical testing and could be helpful to the comparison of results from different laboratories.https://www.frontiersin.org/article/10.3389/fonc.2020.558106/fullexosomesexosomal DNAmutation detectioncancermethodology |
| spellingShingle | Zhe-Ying Wang Rui-Xian Wang Xiao-Qing Ding Xuan Zhang Xiao-Rong Pan Jian-Hua Tong A Protocol for Cancer-Related Mutation Detection on Exosomal DNA in Clinical Application Frontiers in Oncology exosomes exosomal DNA mutation detection cancer methodology |
| title | A Protocol for Cancer-Related Mutation Detection on Exosomal DNA in Clinical Application |
| title_full | A Protocol for Cancer-Related Mutation Detection on Exosomal DNA in Clinical Application |
| title_fullStr | A Protocol for Cancer-Related Mutation Detection on Exosomal DNA in Clinical Application |
| title_full_unstemmed | A Protocol for Cancer-Related Mutation Detection on Exosomal DNA in Clinical Application |
| title_short | A Protocol for Cancer-Related Mutation Detection on Exosomal DNA in Clinical Application |
| title_sort | protocol for cancer related mutation detection on exosomal dna in clinical application |
| topic | exosomes exosomal DNA mutation detection cancer methodology |
| url | https://www.frontiersin.org/article/10.3389/fonc.2020.558106/full |
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