To evaluate potential impacts of cfDNA extraction procedures on total extraction yield and the detection of mutant alleles, plasma samples of a KRAS c.38G > A (10% VAF) positive CRC patient were processed using different manual protocols from various vendors: A Jena PME free-circulating DNA extraction kit (spin-based, carrier RNA: optional) (Analytik Jena, Jena, Germany), QIAamp Circulating NA Kit (vacuum-based, carrier RNA: yes) (Qiagen, Hilden, Germany), and Zymo Quick cfDNA serum and plasma kit (spin-based, carrier RNA: no) (Zymo Research, Irvine, CA, United States). In addition, cfDNA of matched plasma samples from CRC patients (n = 15) was extracted in parallel using the manual protocol by Zymo Research and an automated procedure on a QIAsymphony instrument (Qiagen) using the PAXcircDNA_STA_2400 protocol (Qiagen). All extractions were performed using 1–3 ml of plasma according to the manufacturer’s protocols. For all kits, cfDNA was eluted into 60 μL ddH2O (or TE buffer), quantified by a β-globin–specific qPCR in comparison to a serial dilution of a reference DNA with a known quantity on a 7,500 Real-Time PCR System (Applied Biosystems, Foster City, CA, United States), and stored at −20°C until downstream processing for NGS. All cfDNA concentrations are presented in ng mL−1 plasma to adjust for different volumes of starting material.
Pme free circulating dna extraction kit
Manufactured by Analytik Jena
Sourced in Germany
The PME free-circulating DNA extraction kit is a laboratory equipment product designed to extract and purify free-circulating DNA from various biological samples. The kit utilizes a proprietary technology to efficiently isolate and concentrate DNA fragments without the need for additional processing steps.
Automatically generated - may contain errors
Lab products found in correlation
Qiamp dna blood mini kit, by Qiagen (2 mentions)
Nanodrop nd 1000 spectrometer, by Thermo Fisher Scientific (2 mentions)
Zymo quick cfdna serum and plasma kit, by Zymo Research (1 mentions)
Qiaamp circulating na kit, by Qiagen (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Qiasymphony instrument, by Qiagen (1 mentions)
Agilent high sensitivity dna kit, by Agilent Technologies (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Qiamp circulating nucleic acid kit, by Qiagen (1 mentions)
Nucleospin plasma xs kit, by Macherey-Nagel (1 mentions)
Triton x 100, by Merck Group (1 mentions)
5 protocols using pme free circulating dna extraction kit
1
Evaluating cfDNA Extraction Methods for CRC
2
cfDNA Extraction from Serum and Plasma
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Circulating DNA was separated from 1 ml serum and plasma samples with the PME free-circulating DNA extraction kit (Analytik Jena AG, Jena, Germany), according to the manufacturer's instructions for up to 1-ml extractions with the lysis solution SE/binding solution SBS mechanism. The process consisted of several steps as follows: i) capturing cfDNA in the polymer by mixing serum/plasma with 30 µl of VCR-1 and 150 µl of VCR-2, followed by centrifugation at 18,500 × g for 3 min at room temperature; ii) washing the polymer/DNA complex with 1 ml of ddH2O; iii) adding 400 µl of lysis solution SE to dissolve the pellet; iv) adding 50 µl of PK and incubating the samples for 15 min at 70°C; v) adding 400 µl of binding solution SBS and transferring the samples to spin filters; vi) washing the samples by adding 500 µl of washing solution GS and 650 µl of washing solution BS, followed by centrifugation at 13,500 × g for 1 min at room temperature; and vii) eluting cfDNA in 50 µl of water and storing the cfDNA at −80°C until further analysis. The presence of cfDNA and its fragment size distribution were evaluated by using the Agilent High Sensitivity DNA kit (Agilent Technologies, Inc., Santa Clara, CA, USA) on the 2100 Bioanalyzer.
3
Quantifying Cell-free DNA in Serum and Plasma
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Preoperative blood samples were usually collected on the day prior to RC at a median of 39 days [interquartile range (IQR): 27; 61] after the preceding TURB. Serum and plasma were prepared from 6 ml whole blood. cfDNA was extracted from serum and plasma using diverse DNA extraction kits (i.e., QiAmp DNA Blood Mini kit, Qiagen, Hilden, Germany; QiAmp Circulating Nucleic Acid kit, Qiagen; NucleoSpin Plasma XS kit, Macherey Nagel, Düren, Germany; PME free-circulating DNA Extraction kit, Analytik Jena, Germany). cfDNA was extracted from 2 ml serum or plasma as well as leukocytes (reference) from 6 ml EDTA blood, and performed according to the manufacturer´s instructions. Quantification and quality of the extracted cfDNA were determined spectrophotometrically using the NanoDrop Spectrometer ND-1000 (Thermo Fisher Scientific, Wilmington, DE, USA).
4
Plasma cfDNA Extraction and Quantification
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Blood samples were collected using commercially available EDTA tubes and plasma was extracted and frozen within one hour of collection. Plasma was extracted through two subsequent centrifugation steps at 3000 rpm and 14,000 rpm, each for 10 min at 4 °C. Obtained plasma was stored at − 80 °C until extraction of cfDNA. cfDNA was extracted from 4 ml plasma following the SEP/SBS protocol of the PME-free circulating DNA extraction kit (Analytik Jena, cat. no. 845-IR-0003050), following manufacturer’s instructions. Two subsequent elution steps with each 30 μl Elution Buffer were performed to optimize the yield of extracted cfDNA. DNA was stored at − 20 °C until cfDNA quantification. cfDNA was evaluated with fragment analyzer and quantified using Qubit 2.0 fluorometer. In patients with resectable PDAC, DNA yield from 4 ml of plasma typically ranged from 1 to 20 ng/μl.
5
Preoperative blood sample analysis
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Preoperative blood samples were usually collected on the day prior to RC at a median of 39 days [interquartile range (IQR): 27; 61] after the preceding TURB. Serum was prepared from 6 ml whole blood by 2 centrifugation steps of 3000 g and 16,000 g each for 10 min. Leukocytes (reference) were extracted from 6 ml EDTA blood supplemented up to 50 ml with lysis buffer containing 0.3 M sucrose, 10 mM Tris–HCl pH 7.5, 5 mM MgCl2 and 1% Triton X100 (Sigma, Taufkirchen, Germany). Following incubation for 15 min on ice, the isolation and purification of the leukocytes were carried out by 2 centrifugation steps at 2500 g, at 4 °C for 20 min. cfDNA was extracted from 2 ml serum using the PME free-circulating DNA Extraction kit (Analytik Jena), while DNA was extracted from leukocytes using the Qiamp DNA Blood Mini kit (Qiagen, Hilden, Germany). These DNA extractions were carried out according to the manufacturer´s instructions and similar to the procedure as described above. Quantification and quality of the extracted cfDNA were determined spectrophotometrically using the NanoDrop Spectrometer ND-1000 (Thermo Fisher Scientific, Wilmington, DE, USA).
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