Original scientific paper
The Most Favourable Procedure for the Isolation of Cell-free DNA from the Plasma of Iso-immunized RHD-negative Pregnant Women
Riyaz Ahmad Rather
; Department of Obstetrics and Gynaecology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India; Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research (PGIMER), Cha
Subhas Chandra Saha
; Department of Obstetrics and Gynaecology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India
Veena Dhawan
; Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, India
Abstract
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.
Keywords
Cell-free Foetal DNA; Plasma; Iso-immunization; Genome Equivalents; Elution Volume
Hrčak ID:
161457
URI
Publication date:
1.1.2015.
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