Evaluation of variability in cell-free DNA extraction efficiency from plasma and urine and spike-in normalization
Abstract Cell-free DNA (cfDNA) is a promising new biomarker for clinical use in e.g. oncology and transplantation medicine. Urinary cfDNA is also gaining interest as a non-invasive biomarker. However, cfDNA extraction is not standardised, leading to a variety of different methods being used with var...
Saved in:
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2025-07-01
|
| Series: | Scientific Reports |
| Subjects: | |
| Online Access: | https://doi.org/10.1038/s41598-025-06563-z |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849334562537078784 |
|---|---|
| author | Fanny Sandberg Nicholas Kueng Carlo Rodolfo Largiadèr Ursula Amstutz |
| author_facet | Fanny Sandberg Nicholas Kueng Carlo Rodolfo Largiadèr Ursula Amstutz |
| author_sort | Fanny Sandberg |
| collection | DOAJ |
| description | Abstract Cell-free DNA (cfDNA) is a promising new biomarker for clinical use in e.g. oncology and transplantation medicine. Urinary cfDNA is also gaining interest as a non-invasive biomarker. However, cfDNA extraction is not standardised, leading to a variety of different methods being used with varying efficiencies and size-specificities. In this study, we aimed to assess the variability in cfDNA extraction efficiency for multiple cfDNA extraction methods (QIAamp Circulating Nucleic Acid Kit, Zymo Quick-DNA Urine Kit, Q Sepharose protocol (Qseph)) using the artificial spike-in CEREBIS and compare the contribution of variable extraction efficiency to overall variability in cfDNA quantities determined using droplet digital PCR (ddPCR). We found reproducible extraction efficiencies specific for each method with 84.1% (± 8.17) in plasma, 58.7% (± 11.1) for Zymo, as well as 30.2% (± 13.2) for Qseph based on the 180 bp CEREBIS (Construct to Evaluate the Recovery Efficiency of cfDNA extraction and BISulphite modification) spike-in. Additionally, while the largest proportion of the technical variability was observed between extractions, it was almost negligible compared to the biological variability. Normalization of urinary cfDNA using creatinine in urine reduced the variability, whereas when normalizing for CEREBIS-based extraction efficiency this was not consistently observed. With overall relatively consistent extraction efficiencies within each cfDNA extraction method, normalization for extraction efficiency using CEREBIS thus did not show a clear benefit but might be considered for comparisons between extraction methods. |
| format | Article |
| id | doaj-art-a1bee26fd109496d8c46abf2a4172dbf |
| institution | Kabale University |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj-art-a1bee26fd109496d8c46abf2a4172dbf2025-08-20T03:45:32ZengNature PortfolioScientific Reports2045-23222025-07-0115111110.1038/s41598-025-06563-zEvaluation of variability in cell-free DNA extraction efficiency from plasma and urine and spike-in normalizationFanny Sandberg0Nicholas Kueng1Carlo Rodolfo Largiadèr2Ursula Amstutz3Graduate School for Cellular and Biomedical Sciences, University of BernGraduate School for Cellular and Biomedical Sciences, University of BernDepartment of Clinical Chemistry, Bern University Hospital, University of BernDepartment of Clinical Chemistry, Bern University Hospital, University of BernAbstract Cell-free DNA (cfDNA) is a promising new biomarker for clinical use in e.g. oncology and transplantation medicine. Urinary cfDNA is also gaining interest as a non-invasive biomarker. However, cfDNA extraction is not standardised, leading to a variety of different methods being used with varying efficiencies and size-specificities. In this study, we aimed to assess the variability in cfDNA extraction efficiency for multiple cfDNA extraction methods (QIAamp Circulating Nucleic Acid Kit, Zymo Quick-DNA Urine Kit, Q Sepharose protocol (Qseph)) using the artificial spike-in CEREBIS and compare the contribution of variable extraction efficiency to overall variability in cfDNA quantities determined using droplet digital PCR (ddPCR). We found reproducible extraction efficiencies specific for each method with 84.1% (± 8.17) in plasma, 58.7% (± 11.1) for Zymo, as well as 30.2% (± 13.2) for Qseph based on the 180 bp CEREBIS (Construct to Evaluate the Recovery Efficiency of cfDNA extraction and BISulphite modification) spike-in. Additionally, while the largest proportion of the technical variability was observed between extractions, it was almost negligible compared to the biological variability. Normalization of urinary cfDNA using creatinine in urine reduced the variability, whereas when normalizing for CEREBIS-based extraction efficiency this was not consistently observed. With overall relatively consistent extraction efficiencies within each cfDNA extraction method, normalization for extraction efficiency using CEREBIS thus did not show a clear benefit but might be considered for comparisons between extraction methods.https://doi.org/10.1038/s41598-025-06563-zCell-Free DNAExtraction efficiencySpike-inLiquid biopsyPre-analytics |
| spellingShingle | Fanny Sandberg Nicholas Kueng Carlo Rodolfo Largiadèr Ursula Amstutz Evaluation of variability in cell-free DNA extraction efficiency from plasma and urine and spike-in normalization Scientific Reports Cell-Free DNA Extraction efficiency Spike-in Liquid biopsy Pre-analytics |
| title | Evaluation of variability in cell-free DNA extraction efficiency from plasma and urine and spike-in normalization |
| title_full | Evaluation of variability in cell-free DNA extraction efficiency from plasma and urine and spike-in normalization |
| title_fullStr | Evaluation of variability in cell-free DNA extraction efficiency from plasma and urine and spike-in normalization |
| title_full_unstemmed | Evaluation of variability in cell-free DNA extraction efficiency from plasma and urine and spike-in normalization |
| title_short | Evaluation of variability in cell-free DNA extraction efficiency from plasma and urine and spike-in normalization |
| title_sort | evaluation of variability in cell free dna extraction efficiency from plasma and urine and spike in normalization |
| topic | Cell-Free DNA Extraction efficiency Spike-in Liquid biopsy Pre-analytics |
| url | https://doi.org/10.1038/s41598-025-06563-z |
| work_keys_str_mv | AT fannysandberg evaluationofvariabilityincellfreednaextractionefficiencyfromplasmaandurineandspikeinnormalization AT nicholaskueng evaluationofvariabilityincellfreednaextractionefficiencyfromplasmaandurineandspikeinnormalization AT carlorodolfolargiader evaluationofvariabilityincellfreednaextractionefficiencyfromplasmaandurineandspikeinnormalization AT ursulaamstutz evaluationofvariabilityincellfreednaextractionefficiencyfromplasmaandurineandspikeinnormalization |