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

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.

Investigation of plasma cell-free DNA as a potential biomarker in horses

“…Protocols for sample processing and storage conditions were based on published research in humans and dogs, as well as our own preliminary data. 2,8,19,20 DNA was extracted from plasma using a commercial kit (DNeasy blood and tissue kit; Qiagen) following a modified 18 manufacturer's protocol. Concentrations of cfDNA in thawed plasma and extracted-plasma samples were measured (Qubit 4 benchtop fluorometer, Qubit 1× dsDNA HS assay kit, Invitrogen; Thermo Fisher), in accordance with the manufacturer's directions and as reported previously for canine and human plasma 3,6,10,21,23 and human extractedplasma DNA samples.…”

Non-invasive prenatal rhesus D genotyping using cell-free foetal DNA

“…The DNA was finally eluted with 30 μl of elution buffer. RHD gene sequence detection was performed as described elsewhere91011 with slight modification in PCR reaction conditions. The human β-globin gene was used as a positive control for total DNA extraction, and the plasma obtained from the nulligravida women carrying RhD-positive or RhD-negative blood was used as positive and negative control, respectively.…”

“…PCR conditions were as follows: An initial denaturation step of 10 min at 95°C, followed by amplification performed for 40 cycles of denaturation (95°C for 15 sec), annealing (58°C for 1 min) and extension (72°C for 30 sec). Melting curve analysis was carried out at the end of each PCR assay to verify the specificities of the amplified product11. Each sample was run in triplicate, and for each run, plasma obtained from nulligravida women carrying an RhD-positive or RhD-negative blood was used as positive and negative control, respectively.…”

Techniques of using circulating tumor DNA as a liquid biopsy component in cancer management

“…In order to extract cfDNA from the blood, different methods have been developed. Magnetic enrichment of cfDNA can be achieved by positively charged magnetic beads that bind the negatively charged phosphate backbone of DNA [ [38] , [39] , [40] , [41] ], whereas silica column-based enrichment makes use of the binding affinity of DNA molecules [ [38] , [39] , [40] , [42] , [43] , [44] ]. Furthermore, cfDNA capturing can be performed by polymer mediated enrichment (PME) [ 39 ] or by a phenol-chloroform based extraction procedure in which DNA is not soluble [ 42 ].…”