The Most Favourable Procedure for the Isolation of Cell-Free DNA from the Plasma of Iso-Immunized RHD-Negative Pregnant Women

Background: The ability to achieve quality recovery of cell-free foetal DNA is important for making non-invasive prenatal diagnoses. In this study, we performed quantitative and qualitative analyses of isolated DNA from maternal plasma, using different DNA-isolation methods. Method: DNA was isolated from 30 iso-immunized women via the QIAamp column-based method, using four different elution volumes and two conventionally based methods. Real-time polymerase chain-reaction quantification of RHD and β-globin genes was performed in order to determine foetal-specific sequences and total genome equivalents, respectively. Results: The column-based method at a 3 μl elution volume yielded the highest quality and quantity of total DNA (67.0±0.6 ng/μL). At a 3 μl elution volume, the β-globin and RHD ‐gene sequences were estimated to be the highest among all isolation procedures, with 2778.13±1.5 and 66.9±0.6 GEq/mL, respectively, and a 100% sensitivity for RHD ‐gene sequence detection. Among the two conventional manual methods, the boiling lysis method yielded a higher DNA concentration (53.8±0.8 ng/μL) and purity (1.73±0.05). In addition, the method's sensitivity for foetal-detection sequences was only 80%, whereas the salting-out method's sensitivity was just 70%. Conclusions: This study confirms the theory that the QIAamp method is a specific and sensitive approach for purifying and quantifying plasma DNA, when used in the minimum elution volume.