Evaluation of extraction methods for methylated cell-free fetal DNA from maternal plasma

Lim, Ji Hyae; Lee, Bom Yi; Kim, Jin Woo; Han, You Jung; Chung, Jin Hoon; Kim, Min Hyoung; Kwak, Dong Wook; Park, So Yeon; Choi, Hee Back; Ryu, Hyun Mee

doi:10.1007/s10815-018-1114-8

Cited by 8 publications

(8 citation statements)

References 21 publications

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“…In contrast, in this study, the methylated DNA quantity extracted by NPM kit was significantly lower than other tested protocol. This is consistent with findings of Lim et al, who showed that although the magnetic bead kit is cheaper and quicker to use than the DSP kit, it is not suitable for extracting low copies of methylated cffDNA from maternal plasma for downstream analyses [24].…”

Section: Discussionsupporting

confidence: 92%

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Evaluation and statistical optimization of a method for methylated cell-free fetal DNA extraction from maternal plasma

Akbariqomi

Heidari

Gargari

et al. 2019

J Assist Reprod Genet

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Purpose Methylated cell-free fetal DNA (cffDNA) in maternal plasma can potentially be used as a biomarker for accurate noninvasive prenatal testing (NIPT) of fetal disorders. Recovery and purification of cffDNA are key steps for downstream applications. In this study, we aimed to developed and evaluated different aspects of an optimized method and compared its efficiency with common methods used for extraction of methylated cffDNA. Methods Single factor experiments, Plackett-Burman (PB) design, and response surface methodology (RSM) were conducted for conventional Triton/Heat/Phenol (cTHP) method optimization. The total cell-free DNA (cfDNA) was extracted from pooled maternal plasma using the optimized method called the Triton/Heat/Phenol/Glycogen (THPG), cTHP method, a column-based kit, and a magnetic bead-based kit. In the next step, methylated cfDNA from the extracted total cfDNA was enriched using a methylated DNA immunoprecipitation (MeDIP) kit. Real-time quantitative polymerase chain reaction was performed on the RASSF1 gene and hyper region to determine the genomic equivalents per milliliter (GEq/ml) values of the methylated cfDNA and cffDNA, respectively. Results The optimum values of the significant factors affecting cfDNA extraction from 200 μl of plasma were 3% SDS, 1% Triton X-100, 0.9 μg/μl glycogen, and 0.3 M sodium acetate. The GEq/ml values of methylated cffDNA extracted using the THPG method were significantly higher than for the tested extraction methods (p < 0.001). Mostafa Akbariqomi and Reza Heidari contributed equally to this work.

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Section: Discussionsupporting

confidence: 92%

“…Recent studies have tested the performance of cTHP method, the DSP, and NPM kits for extracting cffDNA from plasma [14,18,23,24]. Xue et al showed that the cTHP method has high efficiency to extract the short DNA fragments smaller than 100 bp [18].…”

Section: Discussionmentioning

confidence: 99%

Evaluation and statistical optimization of a method for methylated cell-free fetal DNA extraction from maternal plasma

Akbariqomi

Heidari

Gargari

et al. 2019

J Assist Reprod Genet

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“…In this study, our results indicated that delaying the time of NIPT blood collection significantly decreased (about 10%) the mean fetal fraction differences between the TG groups, especially in pregnant women with elevated TG (>2.30 mmol/L) levels. It is more likely that apoptotic placental cells (trophoblasts) are a major source of fetal-derived cfDNA in maternal plasma ( 32 ), and the number of apoptotic placental cells is proportional to the mass. Therefore, as the interval increases, the weight of the placenta increases and its apoptotic release increases.…”

Section: Discussionmentioning

confidence: 99%

Lipid Metabolism Affects Fetal Fraction and Screen Failures in Non-invasive Prenatal Testing

et al. 2022

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Objective: To assess the association between lipid metabolism and fetal fraction, which is a critical factor in ensuring a highly accurate non-invasive prenatal testing (NIPT), and on the rate of screen failures or “no calls” in NIPT.Methods: A total of 4,514 pregnant women at 12–26 weeks of gestation underwent NIPT sequencing and serum lipid measurements. Univariate analysis and multivariate regression models were used to evaluate the associations of serum lipid concentrations with the fetal fraction and the rate of screen failures.Results: The fetal fraction decreased with increased low-density lipoprotein cholesterol and triglyceride (TG) levels, which were significant factors (standardized coefficient: −0.11). Conversely, high-density lipoprotein cholesterol and the interval between the two tests were positively correlated with the fetal fraction. The median fetal fraction was 10.88% (interquartile range, 8.28–13.89%) and this decreased with TG from 11.56% at ≤1.10 mmol/L to 9.51% at >2.30 mmol/L. Meanwhile, multivariate logistic regression analysis revealed that increased TG levels were independently associated with the risk of screen failures. The rate of screen failures showed an increase with TG levels from 1.20% at ≤1.70 mmol/L to 2.41% at >2.30 mmol/L.Conclusions: The fetal fraction and the rate of screen failures in NIPT are affected by TG levels. Meanwhile, in pregnant women with high TG levels, delaying the time between NIPT blood collections can significantly increase the fetal fraction.

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“…Increasing use of the cfDNA-based NIPT has created unprecedented challenges in automation in the biotechnology industry. 5 Current cfDNA extraction methods 8,9 mainly include: (I) column-based methods, such as the QIAamp circulating nucleic acid kit [10][11][12][13][14] ; (II) magnetic bead-based methods, such as the NextPrep-Mag™ cfDNA isolation kit 13 ; (III) polymer-mediated enrichment, such as the PME free-circulating DNA extraction kit. 13 However, the preprocessing separation of serum or plasma from whole blood (WB) samples limits the performance of the above cfDNA extraction methods.…”

Section: Introductionmentioning

confidence: 99%

“…Pre-processing is a time-consuming method that requires high-speed centrifugation at low or room temperature for 15-30 min, while avoiding contamination of the blood cells during pipetting operations. 2,9,14 The main challenge faced by many engineers is the automation of the separation of serum or plasma costeffectively. This includes intelligent control of the centrifuge for automatic tube insertion, removal, identification of plasma and buffy coat, shorter (<30 min) separation operations, reduced cost, and increased throughput stability.…”

Section: Introductionmentioning

confidence: 99%

A novel method for extracting circulating cell‐free DNA from whole blood samples and its utility in the non‐invasive prenatal test

et al. 2022

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Objective We verified a magnetic bead‐based, simple, and fast method for circulating cell‐free DNA (cfDNA) extraction from whole blood samples(CEWB) and characterised its utility in non‐invasive prenatal testing (NIPT). Method We extracted cfDNA from both plasma and whole blood of the patients using CEWB and compared it to that extracted using a Qiagen extraction kit; droplet digital polymerase chain reaction test was used to calculate the fragment size bias. In all, 304 samples were used for NIPT. Results The CEWB group (mean ± standard deviation [SD]: 4.34 ± 0.41 ng/ml plasma) reported less DNA weight yield than the Qiagen group (4.90 ± 0.50 ng/ml plasma). There was no significant difference between the CEWB group and the Qiagen group in the gene fragments (136 bp: p = 0.064 and 420 bp: p = 0.534). In a parallel cohort study to characterise the utility of the CEWB method in NIPT, the treatment group extracted by CEWB showed a sensitivity of 100%, a specificity of 99.65%, and a positive predictive value of 95%. Conclusions This study demonstrated that CEWB achieves an acceptable yield of DNA without contamination from genomic DNA. Subsequent clinical experiments in a parallel cohort indicated its utility for NIPT.

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Evaluation of extraction methods for methylated cell-free fetal DNA from maternal plasma

Abstract: Our findings demonstrate that the column-based kit was more effective than the magnetic bead-based kit for isolation of methylated FSERs from maternal plasma as assessed by FSER detection.

Cited by 8 publications

References 21 publications

Evaluation and statistical optimization of a method for methylated cell-free fetal DNA extraction from maternal plasma

Evaluation and statistical optimization of a method for methylated cell-free fetal DNA extraction from maternal plasma

Lipid Metabolism Affects Fetal Fraction and Screen Failures in Non-invasive Prenatal Testing

A novel method for extracting circulating cell‐free DNA from whole blood samples and its utility in the non‐invasive prenatal test

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