Sandberg Fanny, Kueng Nicholas, Largiadèr Carlo Rodolfo, Amstutz Ursula
Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.
Department of Clinical Chemistry, Bern University Hospital, University of Bern, Bern, Switzerland.
Sci Rep. 2025 Jul 2;15(1):22999. doi: 10.1038/s41598-025-06563-z.
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.
游离DNA（cfDNA）是一种很有前景的新型生物标志物，可用于肿瘤学和移植医学等临床领域。尿cfDNA作为一种非侵入性生物标志物也越来越受到关注。然而，cfDNA提取尚未标准化，导致使用了各种不同的方法，其效率和大小特异性各不相同。在本研究中，我们旨在使用人工掺入的CEREBIS评估多种cfDNA提取方法（QIAamp循环核酸试剂盒、Zymo Quick-DNA尿液试剂盒、Q Sepharose方案（Qseph））的cfDNA提取效率的变异性，并比较可变提取效率对使用液滴数字PCR（ddPCR）测定的cfDNA量的总体变异性的贡献。我们发现每种方法都有可重复的提取效率，基于180 bp的CEREBIS（评估cfDNA提取和亚硫酸氢盐修饰回收效率的构建体）掺入，血浆中的提取效率为84.1%（±8.17），Zymo为58.7%（±11.1），Qseph为30.2%（±13.2）。此外，虽然在提取之间观察到最大比例的技术变异性，但与生物变异性相比几乎可以忽略不计。使用尿肌酐对尿cfDNA进行标准化可降低变异性，而在基于CEREBIS的提取效率进行标准化时，并未始终观察到这种情况。由于每种cfDNA提取方法内的提取效率总体相对一致，因此使用CEREBIS对提取效率进行标准化并未显示出明显的益处，但在比较提取方法时可能会考虑。
