Size and Methylation Index of Cell-Free and Cell-Surface-Bound DNA in Blood of Breast Cancer Patients in the Contest of Liquid Biopsy
Aberrantly methylated circulating DNA (cirDNA) has proven to be a good cancer marker, but its detection is limited by low concentrations, fragmentation, and insufficiency. Since the methylated cirDNA was shown to be more stable in circulation than the unmethylated one and was shown to bind with the...
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MDPI AG
2022-08-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/16/8919 |
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| author | Svetlana Tamkovich Alexey Tupikin Anton Kozyakov Pavel Laktionov |
| author_facet | Svetlana Tamkovich Alexey Tupikin Anton Kozyakov Pavel Laktionov |
| author_sort | Svetlana Tamkovich |
| collection | DOAJ |
| description | Aberrantly methylated circulating DNA (cirDNA) has proven to be a good cancer marker, but its detection is limited by low concentrations, fragmentation, and insufficiency. Since the methylated cirDNA was shown to be more stable in circulation than the unmethylated one and was shown to bind with the blood cell surface, we studied the concentration, representation, and fragmentation of tumor-derived methylated DNA in cell-free and cell-surface-associated DNA. We found that long DNA fragments (more than 10 kb) are mainly associated with the surface of blood cells. However, in plasma short DNA fragments (100–1000 bp) were also found along with long DNA fragments. Isolation of short fragments after separation of cirDNA in 6% PAGE followed by quantitative PCR (L1 element) has shown that short DNA fragments in healthy females represent 22% versus 0.5–4.4% in breast cancer patients. The methylated form of the <i>RARβ2</i> gene was detected only in long DNA fragments by Real-time TaqMan PCR of bisulfite-converted DNA. The methylation index of cirDNA from healthy women was estimated at 0%, 9%, and 7% in plasma, PBS-EDTA, and trypsin eluates from the surface of blood cells, respectively. The methylation index of breast cancer patients’ DNA was found to be 33%, 15%, and 61% in the same fractions confirming the overrepresentation of methylated DNA in csbDNA. |
| first_indexed | 2024-03-09T04:21:18Z |
| format | Article |
| id | doaj.art-c9fd448812484c919ef219d8950acd63 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T04:21:18Z |
| publishDate | 2022-08-01 |
| publisher | MDPI AG |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-c9fd448812484c919ef219d8950acd632023-12-03T13:47:12ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-08-012316891910.3390/ijms23168919Size and Methylation Index of Cell-Free and Cell-Surface-Bound DNA in Blood of Breast Cancer Patients in the Contest of Liquid BiopsySvetlana Tamkovich0Alexey Tupikin1Anton Kozyakov2Pavel Laktionov3Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, Novosibirsk 630090, RussiaInstitute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, Novosibirsk 630090, RussiaDepartment of Mammology, Novosibirsk Regional Clinical Oncological Dispensary, Novosibirsk 630108, RussiaInstitute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, Novosibirsk 630090, RussiaAberrantly methylated circulating DNA (cirDNA) has proven to be a good cancer marker, but its detection is limited by low concentrations, fragmentation, and insufficiency. Since the methylated cirDNA was shown to be more stable in circulation than the unmethylated one and was shown to bind with the blood cell surface, we studied the concentration, representation, and fragmentation of tumor-derived methylated DNA in cell-free and cell-surface-associated DNA. We found that long DNA fragments (more than 10 kb) are mainly associated with the surface of blood cells. However, in plasma short DNA fragments (100–1000 bp) were also found along with long DNA fragments. Isolation of short fragments after separation of cirDNA in 6% PAGE followed by quantitative PCR (L1 element) has shown that short DNA fragments in healthy females represent 22% versus 0.5–4.4% in breast cancer patients. The methylated form of the <i>RARβ2</i> gene was detected only in long DNA fragments by Real-time TaqMan PCR of bisulfite-converted DNA. The methylation index of cirDNA from healthy women was estimated at 0%, 9%, and 7% in plasma, PBS-EDTA, and trypsin eluates from the surface of blood cells, respectively. The methylation index of breast cancer patients’ DNA was found to be 33%, 15%, and 61% in the same fractions confirming the overrepresentation of methylated DNA in csbDNA.https://www.mdpi.com/1422-0067/23/16/8919circulating DNAcell-free DNAcell surface bound DNAfragment sizesmethylationbreast cancer |
| spellingShingle | Svetlana Tamkovich Alexey Tupikin Anton Kozyakov Pavel Laktionov Size and Methylation Index of Cell-Free and Cell-Surface-Bound DNA in Blood of Breast Cancer Patients in the Contest of Liquid Biopsy International Journal of Molecular Sciences circulating DNA cell-free DNA cell surface bound DNA fragment sizes methylation breast cancer |
| title | Size and Methylation Index of Cell-Free and Cell-Surface-Bound DNA in Blood of Breast Cancer Patients in the Contest of Liquid Biopsy |
| title_full | Size and Methylation Index of Cell-Free and Cell-Surface-Bound DNA in Blood of Breast Cancer Patients in the Contest of Liquid Biopsy |
| title_fullStr | Size and Methylation Index of Cell-Free and Cell-Surface-Bound DNA in Blood of Breast Cancer Patients in the Contest of Liquid Biopsy |
| title_full_unstemmed | Size and Methylation Index of Cell-Free and Cell-Surface-Bound DNA in Blood of Breast Cancer Patients in the Contest of Liquid Biopsy |
| title_short | Size and Methylation Index of Cell-Free and Cell-Surface-Bound DNA in Blood of Breast Cancer Patients in the Contest of Liquid Biopsy |
| title_sort | size and methylation index of cell free and cell surface bound dna in blood of breast cancer patients in the contest of liquid biopsy |
| topic | circulating DNA cell-free DNA cell surface bound DNA fragment sizes methylation breast cancer |
| url | https://www.mdpi.com/1422-0067/23/16/8919 |
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