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

Akbariqomi, M and Heidari, R and Gargari, S and Omrani, M and Rigi, G and Sanikhani, N and Kooshki, H and Mahmoudian, F and Mazlomi, M and Tavoosidana, G (2019) Evaluation and statistical optimization of a method for methylated cell-free fetal DNA extraction from maternal plasma. Journal of Assisted Reproduction and Genetics.


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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). Conclusions: Our results indicate that the THPG method is more efficient than the other tested methods for extraction of low copy number methylated cffDNA from a small volume of maternal plasma

Item Type: Article
Uncontrolled Keywords: cffDNA extraction, Epigenetic markers, Methylated cell-free fetal DNA, Noninvasive prenatal testing, Plasma, THPG method
Subjects: QS Human Anatomy
QS Human Anatomy
university Structure > QS Human Anatomy

QT physiology
WH Hemic and Lymphatic System
Divisions: Faculty of Medicine > Department of Clinical Sciences > Department of Obstetrics and Gynecology
Depositing User: Unnamed user with email nazari@skums.ac.ir
Date Deposited: 07 Apr 2019 05:33
Last Modified: 16 Apr 2019 08:18
URI: http://eprints.skums.ac.ir/id/eprint/7604

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