Evaluation and statistical optimization of a method for methylated cell-free fetal DNA extraction from maternal plasma.
Author(s): Akbariqomi M, Heidari R, Gargari SS, Omrani MD, Rigi G, Sanikhani NS, Kooshki H, Mahmoudian F, Mazlomi MA, Tavoosidana G
Publication: J Assist Reprod Genet, 2019, Vol. , Page
PubMed ID: 30820784 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of optimizing the extraction buffer, digestion, purification and precipitation steps of the Triton/heat/phenol method on the yield of methylated cell-free fetal DNA (cffDNA). The yield of methylated cell-free DNA (cfDNA) and cffDNA obtained with the optimized method was then compared to that obtained using the standard Triton/heat/phenol method, the QIAamp DSP virus kit, and the NextPrep-Mag cfDNA isolation kit.
Conclusion of Paper
The highest yields of methylated cffDNA were obtained when the method included digestion for 2 h at 56˚C, 1% Triton-SDS extraction buffer, phenol-chloroform-isoamyl alcohol purification, and precipitation with ethanol-glycogen sodium acetate. In the optimization, the highest yields were obtained when 1 mL of blood was extracted in a solution containing 1% SDS and 1% Triton X-100, incubated at 95˚C for 5 min, and precipitated for 1 h at -20˚C in a solution containing 1 M sodium acetate pH 5.2, 1 ug/ul glycogen, and 2.5 volumes ethanol. ANOVA analysis revealed significant effects of the percentage of SDS and the concentration of glycogen and sodium acetate. Higher yields of methylated cfDNA and cffDNA were obtained using the optimized method than using the standard Triton/heat/phenol method, the QIAamp DSP virus kit, or the NextPrep-Mag cfDNA isolation kit.
Studies
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Study Purpose
This study investigated the effects of proteinase K digestion protocols, extraction buffers, purification methods, and precipitation agents on the cffDNA yield. Peripheral blood was collected from 35 healthy pregnant women into K3EDTA tubes. Plasma was obtained by immediate centrifugation at 1600 x g for 12 min, aliquoted, and then stored at -80˚C. The cfDNA was extracted using a Triton/heat/phenol protocol. Proteinase K digestion optimization protocols included incubation at 37˚C for 2 h (control), 37˚C for 30 min, 56˚C for 2 h, 56˚C for 30 min, and room temperature overnight. Extraction buffers containing 1% Triton X-100 (control), TNES buffer, sodium iodide, 1% Tween 80, potassium chloride, 1% Triton-SDS, 12 M guanidinium thiocyanate, urea, or sodium perchlorate were compared. cfDNA was purified with phenol-chloroform-isoamyl alcohol (control), phenol-chloroform-isoamyl alcohol with beta mercaptoethanol, TRIzol, or 2% CTAB. cfDNA was precipitated with ethanol-sodium acetate (control), ethanol sodium citrate, ethanol-ammonium acetate, ethanol-glycogen sodium acetate, ethanol-potassium acetate, PEG8000-ethanol-NaCl, or PEG8000-NaCl-MgCl2. cffDNA concentrations were determined by real-time PCR amplification of the RASSF1 gene and a hypermethylated region.
Summary of Findings:
Significantly more methylated cffDNA was obtained when specimens were digested at 56˚C for 2 h rather than 37˚C for 2 h (14% increase, P=0.049); however, digestion at 37˚C or 56˚C for 30 min resulted in significantly lower yield than in specimens digested at 37˚C for 2 h (P<0.001, both). Comparable yields were obtained after digestion at room temperature overnight to those from specimens digested at 37°C for 2 h. Compared to extraction with 1% Triton, significantly more cffDNA was obtained using 1%Triton-SDS (40% increase, P<0.001), sodium iodide (24% increase, P<0.001), and guanidinium thiocyanate (16% increase, P=0.0107) and significantly less methylated cffDNA with potassium chloride (P=0.031), TNES buffer (P<0.001), and urea (P<0.001). Extraction with sodium perchlorate and sodium iodide resulted in comparable yields methylated cffDNA compared to extraction with 1% Triton. Purification with phenol-chloroform-isoamyl alcohol with beta mercaptoethanol, TRIzol, or 2% CTAB all resulted in a decrease in isolated methylated cffDNA compared to purification with only phenol-chloroform isoamyl alcohol (P<0.001, all). Precipitation with ethanol-glycogen sodium acetate yielded more methylated cffDNA than ethanol-sodium acetate (45% increase, P<0.001). Precipitation with ethanol-potassium acetate and ethanol-sodium acetate yielded comparable methylated cffDNA to ethanol-sodium acetate, and all other precipitation agents yielded significantly less methylated cffDNA (P<0.001, all).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method Extraction buffers containing 1% TritonX-100 (control)
Extraction buffers containing TNES buffer
Extraction buffers containing 2 M guanidinium thiocyanate
Extraction buffers containing urea
Extraction buffers containing sodium perchlorate
Extraction buffers containing 1% TritonX-1%SDS
Extraction buffers containing sodium iodide
Extraction buffers containing 1% Tween 80
Extraction buffers containing Potassium chloride
Analyte Extraction and Purification Protein digestion Proteinase K 37˚C for 2 h
Proteinase K 37˚C for 30 min
Proteinase K 56˚C for 2 h
Proteinase K 56˚C for 30 min
Proteinase K room temperature overnight
Analyte Extraction and Purification Analyte purification Phenol-chloroform-isoamyl alcohol (control)
Phenol-chloroform-isoamyl alcohol with betamercaptoethanol
TRIzol
2% CTAB
Precipitated with ethanol-sodium acetate (control)
Precipitated with ethanol sodium citrate
Precipitated with ethanol-ammonium acetate
Precipitated with ethanol-glycogen sodium acetate
Precipitated with ethanol-potassium acetate
Precipitated with PEG8000-Ethanol-NaCl
Precipitated with PEG8000-NaCl-MgCl2
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Study Purpose
This study investigated the effects of plasma volume, concentration of SDS and Triton X-100 in the extraction buffer, incubation temperature, concentration and pH of sodium acetate in the precipitation solution, the presence of glycogen in the precipitation, and the precipitation time on the yield of cffDNA. Peripheral blood was collected from 35 healthy pregnant women into K3EDTA tubes. Plasma was obtained by immediate centrifugation at 1600 x g for 12 min, aliquoted, and then stored at -80˚C. The cfDNA was extracted using a 0 or 1 % SDS with 1 or 2% Triton X-100; and incubated at 85˚C or 90°C for 5 min. cfDNA was precipitated from specimens with a solution containing 1 M sodium acetate (pH 5.2 or 7) and 2.5 volumes of 100% ethanol, and either 0 or 1 μg/μl of glycogen (20 mg/ml) for 1 or 16 h at -20°C. ccfDNA concentrations were determined by real-time PCR amplification of the RASSF1 gene and a hypermethylated region.
Summary of Findings:
The highest yields were obtained when 1 mL of blood extracted in a solution containing 1% SDS and 1% Triton X-100, incubated at 95˚C for 5 min, and precipitated for 1 h at -20˚C in a solution containing 1 M sodium acetate pH 5.2, 1 ug/ul glycogen, and 2.5 volumes of ethanol. ANOVA analysis revealed significant effects of the percentage of SDS (P=0.022), the concentration of glycogen (P<0.001), and sodium acetate (P<0.001). The authors modeled the effects of the percentage of SDS and concentration of glycogen and sodium actetate on the extraction of methylated cffDNA and refined the effects through additional experimentation and construction of response surface and contour graphs.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Aliquot size/volume 200 ul
1 ml
Analyte Extraction and Purification Analyte purification 1 M sodium acetate pH 5 with 1 ug/ul glycogen for 1 h
1 M sodium acetate pH 7 with 1 ug/ul glycogen for 1 h
1 M sodium acetate pH 5 without glycogen for 1 h
1 M sodium acetate pH 7 without glycogen for 1 h
1 M sodium acetate pH 5 with 1 ug/ul glycogen for 16 h
1 M sodium acetate pH 7 with 1 ug/ul glycogen for 16 h
1 M sodium acetate pH 5 without glycogen for 16 h
1 M sodium acetate pH 7 without glycogen for 16 h
Analyte Extraction and Purification Analyte isolation method Extraction buffer containing 0% SDS and 2% Triton
Extraction buffer containing 1% SDS and 2% Triton
Extraction buffer containing 0% SDS and 1% Triton
Extraction buffer containing 1% SDS and 1% Triton
Analyte Extraction and Purification Incubation duration/condition 85˚C
95˚C
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Study Purpose
This study evaluated the effects of extraction method on the yield of methylated cell-free DNA and methylated cell-free fetal DNA from plasma. Peripheral blood was collected from 35 healthy pregnant women into K3EDTA tubes. Plasma was obtained by immediate centrifugation at 1600 x g for 12 min, aliquoted, and then stored at -80˚C. DNA was extracted from pooled plasma using the author’s optimized Triton/heat/phenol/glycogen method, a standard Triton/heat/phenol method, the QIAamp DSP virus kit, or the NextPrep-Mag cfDNA isolation kit. cffDNA concentrations were determined by real-time PCR amplification of the RASSF1 gene and a hypermethylated region.
Summary of Findings:
The highest yields of methylated cfDNA and cffDNA were obtained using the optimized method followed by the standard Triton/heat/phenol method. The NextPrep-Mag cfDNA isolation kit resulted in the lowest yields.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method Optimized Triton/Heat/Phenol/Glycogen method
Standard Triton/Heat/Phenol method
NextPrep-Mag cfDNA isolation kit
QIAamp DSP Virus Kit