Evaluation and statistical optimization of a method for methylated cell-free fetal DNA extraction from maternal plasma.
J Assist Reprod Genet
; 36(5): 1029-1038, 2019 May.
Article
en En
| MEDLINE
| ID: mdl-30820784
ABSTRACT
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.Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Diagnóstico Prenatal
/
Metilación de ADN
/
Técnicas de Diagnóstico Molecular
/
Feto
/
Ácidos Nucleicos Libres de Células
Tipo de estudio:
Diagnostic_studies
/
Evaluation_studies
Límite:
Adult
/
Female
/
Humans
/
Male
/
Middle aged
/
Pregnancy
Idioma:
En
Revista:
J Assist Reprod Genet
Asunto de la revista:
GENETICA
/
MEDICINA REPRODUTIVA
Año:
2019
Tipo del documento:
Article
País de afiliación:
Irán