Evaluation of commercial kits for isolation and bisulfite conversion of circulating cell-free tumor DNA from blood
Abstract Background DNA methylation biomarkers in circulating cell-free DNA (cfDNA) have great clinical potential for cancer management. Most methods for DNA methylation analysis require bisulfite conversion, causing DNA degradation and loss. This is particularly challenging for cfDNA, which is natu...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-09-01
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| Series: | Clinical Epigenetics |
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| Online Access: | https://doi.org/10.1186/s13148-023-01563-0 |
| _version_ | 1797452157367091200 |
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| author | Stine H. Kresse Sara Brandt-Winge Heidi Pharo Bjørnar T. B. Flatin Marine Jeanmougin Hege Marie Vedeld Guro E. Lind |
| author_facet | Stine H. Kresse Sara Brandt-Winge Heidi Pharo Bjørnar T. B. Flatin Marine Jeanmougin Hege Marie Vedeld Guro E. Lind |
| author_sort | Stine H. Kresse |
| collection | DOAJ |
| description | Abstract Background DNA methylation biomarkers in circulating cell-free DNA (cfDNA) have great clinical potential for cancer management. Most methods for DNA methylation analysis require bisulfite conversion, causing DNA degradation and loss. This is particularly challenging for cfDNA, which is naturally fragmented and normally present in low amounts. The aim of the present study was to identify an optimal combination of cfDNA isolation and bisulfite conversion kits for downstream analysis of DNA methylation biomarkers in plasma. Results Of the five tested bisulfite conversion kits (EpiJET Bisulfite Conversion Kit, EpiTect Plus DNA Bisulfite Kit (EpiTect), EZ DNA Methylation-Direct Kit, Imprint DNA Modification Kit (Imprint) and Premium Bisulfite Kit), the highest and lowest DNA yield and recovery were achieved using the EpiTect kit and the Imprint kit, respectively, with more than double the amount of DNA for the EpiTect kit. Of the three tested cfDNA isolation kits (Maxwell RSC ccfDNA Plasma Kit, QIAamp Circulating Nucleic Acid Kit (CNA) and QIAamp MinElute ccfDNA Mini Kit), the CNA kit yielded around twice as much cfDNA compared to the two others kits, although with more high molecular weight DNA present. When comparing various combinations of cfDNA isolation kits and bisulfite conversion kits, the CNA kit and the EpiTect kit were identified as the best-performing combination, resulting in the highest yield of bisulfite converted cfDNA from normal plasma, as measured by droplet digital PCR (ddPCR). As a proof of principle, this kit combination was used to process plasma samples from 13 colorectal cancer patients for subsequent ddPCR methylation analysis of BCAT1 and IKZF1. Methylation of BCAT1 and/or IKZF1 was identified in 6/10 (60%) stage IV patients and 1/3 (33%) stage III patients. Conclusions Based on a thorough evaluation of five bisulfite conversion kits and three cfDNA isolation kits, both individually and in combination, the CNA kit and the EpiTect kit were identified as the best-performing kit combination, with highest DNA yield and recovery across a range of DNA input amounts. The combination was successfully used for detection of clinically relevant DNA methylation biomarkers in plasma from cancer patients. |
| first_indexed | 2024-03-09T15:04:42Z |
| format | Article |
| id | doaj.art-3533e452970e4e5a9487c7ab1707a11a |
| institution | Directory Open Access Journal |
| issn | 1868-7083 |
| language | English |
| last_indexed | 2024-03-09T15:04:42Z |
| publishDate | 2023-09-01 |
| publisher | BMC |
| record_format | Article |
| series | Clinical Epigenetics |
| spelling | doaj.art-3533e452970e4e5a9487c7ab1707a11a2023-11-26T13:41:35ZengBMCClinical Epigenetics1868-70832023-09-0115111410.1186/s13148-023-01563-0Evaluation of commercial kits for isolation and bisulfite conversion of circulating cell-free tumor DNA from bloodStine H. Kresse0Sara Brandt-Winge1Heidi Pharo2Bjørnar T. B. Flatin3Marine Jeanmougin4Hege Marie Vedeld5Guro E. Lind6Department of Molecular Oncology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University HospitalDepartment of Molecular Oncology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University HospitalDepartment of Molecular Oncology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University HospitalDepartment of Molecular Oncology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University HospitalDepartment of Molecular Oncology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University HospitalDepartment of Molecular Oncology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University HospitalDepartment of Molecular Oncology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University HospitalAbstract Background DNA methylation biomarkers in circulating cell-free DNA (cfDNA) have great clinical potential for cancer management. Most methods for DNA methylation analysis require bisulfite conversion, causing DNA degradation and loss. This is particularly challenging for cfDNA, which is naturally fragmented and normally present in low amounts. The aim of the present study was to identify an optimal combination of cfDNA isolation and bisulfite conversion kits for downstream analysis of DNA methylation biomarkers in plasma. Results Of the five tested bisulfite conversion kits (EpiJET Bisulfite Conversion Kit, EpiTect Plus DNA Bisulfite Kit (EpiTect), EZ DNA Methylation-Direct Kit, Imprint DNA Modification Kit (Imprint) and Premium Bisulfite Kit), the highest and lowest DNA yield and recovery were achieved using the EpiTect kit and the Imprint kit, respectively, with more than double the amount of DNA for the EpiTect kit. Of the three tested cfDNA isolation kits (Maxwell RSC ccfDNA Plasma Kit, QIAamp Circulating Nucleic Acid Kit (CNA) and QIAamp MinElute ccfDNA Mini Kit), the CNA kit yielded around twice as much cfDNA compared to the two others kits, although with more high molecular weight DNA present. When comparing various combinations of cfDNA isolation kits and bisulfite conversion kits, the CNA kit and the EpiTect kit were identified as the best-performing combination, resulting in the highest yield of bisulfite converted cfDNA from normal plasma, as measured by droplet digital PCR (ddPCR). As a proof of principle, this kit combination was used to process plasma samples from 13 colorectal cancer patients for subsequent ddPCR methylation analysis of BCAT1 and IKZF1. Methylation of BCAT1 and/or IKZF1 was identified in 6/10 (60%) stage IV patients and 1/3 (33%) stage III patients. Conclusions Based on a thorough evaluation of five bisulfite conversion kits and three cfDNA isolation kits, both individually and in combination, the CNA kit and the EpiTect kit were identified as the best-performing kit combination, with highest DNA yield and recovery across a range of DNA input amounts. The combination was successfully used for detection of clinically relevant DNA methylation biomarkers in plasma from cancer patients.https://doi.org/10.1186/s13148-023-01563-0Liquid biopsyCirculating cell-free tumor DNAcfDNActDNAPlasmacfDNA isolation |
| spellingShingle | Stine H. Kresse Sara Brandt-Winge Heidi Pharo Bjørnar T. B. Flatin Marine Jeanmougin Hege Marie Vedeld Guro E. Lind Evaluation of commercial kits for isolation and bisulfite conversion of circulating cell-free tumor DNA from blood Clinical Epigenetics Liquid biopsy Circulating cell-free tumor DNA cfDNA ctDNA Plasma cfDNA isolation |
| title | Evaluation of commercial kits for isolation and bisulfite conversion of circulating cell-free tumor DNA from blood |
| title_full | Evaluation of commercial kits for isolation and bisulfite conversion of circulating cell-free tumor DNA from blood |
| title_fullStr | Evaluation of commercial kits for isolation and bisulfite conversion of circulating cell-free tumor DNA from blood |
| title_full_unstemmed | Evaluation of commercial kits for isolation and bisulfite conversion of circulating cell-free tumor DNA from blood |
| title_short | Evaluation of commercial kits for isolation and bisulfite conversion of circulating cell-free tumor DNA from blood |
| title_sort | evaluation of commercial kits for isolation and bisulfite conversion of circulating cell free tumor dna from blood |
| topic | Liquid biopsy Circulating cell-free tumor DNA cfDNA ctDNA Plasma cfDNA isolation |
| url | https://doi.org/10.1186/s13148-023-01563-0 |
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