Extraction of cell-free fetal DNA from maternal plasma

Mostafa Akbariqomi, Reza Heidari, Soraya Saleh Gargari, Mir Davood Omrani, Garshasb Rigi, Nafiseh Sadat Sanikhani, Hamid Kooshki, Fatemeh Mahmoudian, Mohammad Ali Mazlomi, Gholamreza Tavoosidana

Overview

Introduction an efficient method (THPG) for Improving the quality and quantity of cell-free fetal DNA (cffDNA) extracted from maternal plasma as an authentic genetic source for use in NIPT of fetal disorders.

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https://pubmed.ncbi.nlm.nih.gov/30820784/
https://link.springer.com/article/10.1007/s10815-019-01425-w

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

J Assist Reprod Genet 2019 May 28;36(5):1029-1038. Epub 2019 Feb 28.

Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.

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 ?l of plasma were 3% SDS, 1% Triton X-100, 0.9 ?g/?l 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?
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.

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http://dx.doi.org/10.1007/s10815-019-01425-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6541686PMC
May 2019
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