To examine the impact of purification kits on the yield of cfDNA, three DNA purification kits were compared: DNeasy Blood & Tissue Kit (Qiagen), QIAamp Circulating Nucleic Acid Kit (Qiagen), and Quick-cfDNA Serum & Plasma Kit (Zymo Research). cfDNA was purified from blood samples collected from healthy volunteers (n = 3). Purification of cfDNA using the QIAamp Circulating Nucleic Acid Kit (Qiagen), Quick-cfDNA Serum & Plasma Kit (Zymo Research), and DNeasy Blood & Tissue Kit (Qiagen) was performed following the manufacturer's instructions. cfDNA was eluted in 50 μl elution buffer. Assessment of cfDNA size and yield was performed by Bioanalyzer and Qubit analysis.
Quick cfdna serum plasma kit
Manufactured by Zymo Research
Sourced in United States
The Quick-cfDNA Serum & Plasma Kit is a laboratory product designed for the rapid extraction of cell-free DNA (cfDNA) from serum and plasma samples. The kit utilizes a proprietary technology to efficiently isolate high-quality cfDNA, which can be used in various downstream applications, such as genetic analysis and biomarker detection.
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21 protocols using quick cfdna serum plasma kit
1
Comparison of cfDNA Purification Kits
2
Circulating cell-free DNA analysis
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cfDNA extraction: Blood samples from HBsAg-positive carriers, patients with liver cirrhosis, and patients with liver cancer were collected at the time of diagnosis and sent to Zhongke Jinzhen Co., Ltd. for DNA extraction, pyrolysis of specimen precipitate after centrifugation, and extraction of cfDNA from plasma using the Quick-cfDNA Serum & Plasma Kit (Zymo).
DNA quality test and results: Qubit accurately quantified the DNA concentration, and Q-sep analyzed the size, distribution, and relative quantification of DNA fragments. After the sample was quantified, the DNA of the sample was first repaired, a tail was added to the 3' end, and the sequencing joint was connected. Biotins were connected by a transglycosylation reaction and click chemistry. The DNA fragment containing 5hmC was captured using streptavidin beads, and PCR amplification was performed to complete the entire library construction. After the library quality inspection, different libraries were sequenced using Nova6000 according to the effective concentration and target data volume. After obtaining the original sequence (sequenced reads), the original gene fragments were returned to the correct human reference genome position to calculate the gene expression of each sample.
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3
cfDNA Isolation from Serum and Media
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Between 200 and 1000 µL of serum and 5 mL conditioned media were available for cfDNA isolation, which was performed with the Quick-cfDNA Serum & Plasma kit (Zymo Research, Irvine, CA, USA), according to manufacturer’s protocol.
4
Comprehensive Genomic DNA Isolation
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Genomic DNA isolation was performed from fresh frozen and formalin-fixed, paraffin-embedded tissue biopsies using a High Pure PCR Template Preparation Kit (Roche) according to the manufacturer’s protocol.
Buffy coat layers were separated from whole blood specimens by centrifugation at 1350 rcf for 12 min. Genomic DNA was isolated from 200 µL buffy coat fractions (High Pure PCR Template Preparation Kit, Roche) in line with the manufacturer’s instructions. In the case of the Cell-Free DNA Collection Tube (Roche), ProtK digestion was performed for one and two hours.
Plasma fractions were separated from whole blood samples by two centrifugation steps (1350 rcf, 12 min). CfDNA isolation was carried out from 1–2 mL plasma samples with the Quick-cfDNA™ Serum & Plasma Kit (Zymo Research, Orange, FL, USA) according to the manufacturer’s recommendation. All DNA samples were stored at −20 °C until further examinations.
A NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Orange, FL, USA) was applied to measure the concentration and purity (OD260/280, OD230/280) of both tissue and buffy coat genomic DNA, while quantification of cfDNA was carried out with a Qubit 1.0 fluorometer using a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific).
Buffy coat layers were separated from whole blood specimens by centrifugation at 1350 rcf for 12 min. Genomic DNA was isolated from 200 µL buffy coat fractions (High Pure PCR Template Preparation Kit, Roche) in line with the manufacturer’s instructions. In the case of the Cell-Free DNA Collection Tube (Roche), ProtK digestion was performed for one and two hours.
Plasma fractions were separated from whole blood samples by two centrifugation steps (1350 rcf, 12 min). CfDNA isolation was carried out from 1–2 mL plasma samples with the Quick-cfDNA™ Serum & Plasma Kit (Zymo Research, Orange, FL, USA) according to the manufacturer’s recommendation. All DNA samples were stored at −20 °C until further examinations.
A NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Orange, FL, USA) was applied to measure the concentration and purity (OD260/280, OD230/280) of both tissue and buffy coat genomic DNA, while quantification of cfDNA was carried out with a Qubit 1.0 fluorometer using a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific).
5
Quantifying Mitochondrial and Nuclear DNA Ratios
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Individual ELISA protocols were performed as per manufacturer’s instructions with the following sample dilutions: For MPO ELISA (abcam cat# ab195212) plasma dilution 1:1000; for C5b-9 ELISA (MyBioSource cat# MBS2021557) plasma dilution 1:100; for IL-6 ELISA (Abcam cat# ab46027) plasma dilution 1:2; for ET-1 ELISA (abcam cat# ab133030) plasma dilution 1:2.
To compare the levels of mitochondrial to nuclear DNA in human plasma samples we used the NovaQUANT™ Human Mitochondrial to Nuclear DNA Ratio Kit (SIGMA-Aldrich cat# 72620-1KIT) as per manufacturer’s instructions. The kit measures the mtDNA copy number to that of nuclear DNA by Real-Time PCR of specific mitochondrial and nuclear genes. Plasma DNA was isolated from 200 uL of plasma using the Quick-cfDNA Serum & Plasma Kit (Zymo Research, cat# D4076) as per manufacturer’s instruction.
To compare the levels of mitochondrial to nuclear DNA in human plasma samples we used the NovaQUANT™ Human Mitochondrial to Nuclear DNA Ratio Kit (SIGMA-Aldrich cat# 72620-1KIT) as per manufacturer’s instructions. The kit measures the mtDNA copy number to that of nuclear DNA by Real-Time PCR of specific mitochondrial and nuclear genes. Plasma DNA was isolated from 200 uL of plasma using the Quick-cfDNA Serum & Plasma Kit (Zymo Research, cat# D4076) as per manufacturer’s instruction.
6
Isolation and Characterization of cfDNA
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CfDNA from the 2 ml of retrospectively collected serum from each patient was isolated using Quick-cfDNA™ Serum & Plasma Kit (Zymo Research) following the manufacturer’s protocol. The DNA was eluted in 25 μl of elution buffer and the quantification was done using Qubit kit on a Qubit fluorometer (Thermo Fisher). An estimated 1 ng/μl of sample were loaded on High Sensitivity DNA chip (Agilent) and run on a Bioanalyzer 2100 (Agilent). The DNA integrity and purity were evaluated. The electrographs were generated using the 2100 expert software (Agilent).
7
Plasma cfDNA Extraction Protocol
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Peripheral blood from patients and NCA individuals was collected for cfDNA preparation. Briefly, 8 ml of peripheral blood was collected into Cell-Free DNA Collection Tubes (Roche). Within 4 h, plasma was prepared by centrifuging twice at 1350×g for 12 min at 4 °C and 13,500×g for 12 min at 4 °C. cfDNA was extracted using the Quick-cfDNA Serum & Plasma Kit (ZYMO) and then stored at − 80 °C. The fragment size of all the cfDNA samples was verified by nucleic acid electrophoresis before library preparation.
8
Genomic and Cell-free DNA Isolation
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Genomic DNA was isolated from FFT biopsies with High Pure PCR Template Preparation Kit (Roche) according to the manufacturer’s instruction with overnight proteinase K digestion.
Plasma fractions were separated from whole blood samples by two centrifugation steps (1350 rcf, 12 min). CfDNA isolation was carried out from 3.5 ml plasma samples with Quick-cfDNA™ Serum & Plasma Kit (Zymo Research) according to the manufacturer's recommendation. DNA samples were stored at -20 °C until further examinations. Concentration and purity (OD260/280, OD230/280) of genomic DNA were measured by NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific), while cfDNA quantification was performed with Qubit 1.0 fluorometer using Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific).
Plasma fractions were separated from whole blood samples by two centrifugation steps (1350 rcf, 12 min). CfDNA isolation was carried out from 3.5 ml plasma samples with Quick-cfDNA™ Serum & Plasma Kit (Zymo Research) according to the manufacturer's recommendation. DNA samples were stored at -20 °C until further examinations. Concentration and purity (OD260/280, OD230/280) of genomic DNA were measured by NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific), while cfDNA quantification was performed with Qubit 1.0 fluorometer using Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific).
9
Quantifying Mitochondrial-to-Nuclear DNA Ratio
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Individual ELISA protocols were performed as per manufacturer's instructions with the following sample dilutions: for MPO ELISA (Abcam cat# ab195212) plasma dilution 1:1,000; for C5b-9 ELISA (MyBioSource cat# MBS2021557) plasma dilution 1:100; for IL-6 ELISA (Abcam cat# ab46027) plasma dilution 1:2; and for ET-1 ELISA (Abcam cat# ab133030) plasma dilution 1:2.
To compare the levels of mitochondrial to nuclear DNA in human plasma samples, we used the NovaQUANT™ Human Mitochondrial to Nuclear DNA Ratio Kit (SIGMA-Aldrich cat# 72620-1KIT) as per the manufacturer's instructions. The kit measures the mtDNA copy number to that of nuclear DNA by Real-Time PCR of specific mitochondrial and nuclear genes optimized for equivalent amplification. Plasma DNA was isolated from 200 μl of plasma using the Quick-cfDNA Serum & Plasma Kit (Zymo Research, cat# D4076) as per the manufacturer's instruction.
To compare the levels of mitochondrial to nuclear DNA in human plasma samples, we used the NovaQUANT™ Human Mitochondrial to Nuclear DNA Ratio Kit (SIGMA-Aldrich cat# 72620-1KIT) as per the manufacturer's instructions. The kit measures the mtDNA copy number to that of nuclear DNA by Real-Time PCR of specific mitochondrial and nuclear genes optimized for equivalent amplification. Plasma DNA was isolated from 200 μl of plasma using the Quick-cfDNA Serum & Plasma Kit (Zymo Research, cat# D4076) as per the manufacturer's instruction.
10
cfDNA Isolation and Bisulfite Conversion
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CfDNA was isolated from 4 mL plasma samples using Quick-cfDNA Serum & Plasma Kit (Zymo Research, Irvine, CA, USA), according to the manufacturer’s instructions, and it was eluted in 50 µL Elution Buffer. The sample concentration was measured with Qubit 1.0 fluorometer using Qubit dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The bisulfite conversion of 50 ng cfDNA was performed using EZ DNA Methylation Direct Kit (Zymo Research) according to the manufacturer’s instructions with the elution volume of 20 µL.
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