Peripheral blood samples were collected in ethylenediaminetetraacetic acid (EDTA) tubes immediately before the first dose of pembrolizumab and every three weeks afterward. The blood samples were centrifuged at 1,400 g for fifteen minutes, and the plasma and buffy coats were isolated and frozen in separate aliquots at −80 ℃. Plasma samples taken before treatment initiation and after two cycles of treatment were chosen for analysis to ensure enough time for an effect of the treatment. Twenty-one patients had no sample available after two cycles of treatment, however, 6/21 patients had a sample collected after one cycle which was included instead. The cfDNA was isolated from the available plasma (2.00–7.30 mL) using AVENIO cfDNA Isolation Kit (Roche, Basel, Switzerland) and eluted in 65 µL elution buffer. DNA was isolated from 0.20 mL buffy coat using AVENIO Tumor DNA Isolation and QC Kit (Roche) and AVENIO Tumor Cleanup and Capture Beads (Roche) and eluted in 30 µL elution buffer. The quality of the isolated cfDNA and DNA was analyzed using QubitTM dsDNA HS assay kit (Thermo Fischer Scientific, Waltham, MA, USA) and 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA).
Avenio cfdna isolation kit
Manufactured by Roche
Sourced in Switzerland, Germany
The AVENIO cfDNA Isolation Kit is a laboratory equipment product designed for the extraction and purification of cell-free DNA (cfDNA) from various biological samples. The kit utilizes a column-based method to isolate high-quality cfDNA, which can be used for downstream applications such as genetic analysis and biomarker detection.
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Lab products found in correlation
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (5 mentions)
Cell free dna collection tube, by Roche (4 mentions)
Picogreen dsdna assay kit, by Thermo Fisher Scientific (4 mentions)
Qubit dsdna high sensitivity kit, by Thermo Fisher Scientific (3 mentions)
2100 bioanalyzer high sensitivity dna kit, by Agilent Technologies (3 mentions)
Nanodrop 2000 device, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Avenio ctdna expanded kit, by Roche (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Qubittm dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Qubit fluorometer 2, by Thermo Fisher Scientific (1 mentions)
Allprep dna rna mini kit, by Qiagen (1 mentions)
Agilent high sensitivity dna kit, by Agilent Technologies (1 mentions)
High sensitivity kit, by Agilent Technologies (1 mentions)
Qubit dsdna hs assay kit with qubit 3.0 fluorimeter, by Thermo Fisher Scientific (1 mentions)
Agilent bioanalyzer, by Agilent Technologies (1 mentions)
Qiaamp minelute ccfdna kit, by Qiagen (1 mentions)
Agilent high sensitivity dna analysis kit, by Agilent Technologies (1 mentions)
Nextseq sequencing platform, by Illumina (1 mentions)
17 protocols using avenio cfdna isolation kit
1
Plasma and Buffy Coat Analysis for Pembrolizumab Response
2
Plasma ctDNA Extraction and Quantification
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Samples (8.5 ml) of peripheral blood were collected from the patients in cell-free DNA collection tubes (Roche Diagnostics, Indianapolis, IN, USA). Plasma ctDNA was purified using an AVENIO cfDNA isolation kit (Roche Diagnostics), according to the manufacturer's instructions. The quality and quantity of the DNA were verified using the NanoDrop 2000 device (Thermo Fisher Scientific, Inc.) and PicoGreen dsDNA assay kit (Thermo Fisher Scientific, Inc.). The extracted ctDNA was stored at −80°C until the analysis.
3
Plasma cfDNA Extraction and Quantification
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Plasma was isolated from whole blood samples by centrifuging at 500× g for 10 min and stored at −80 °C until further use. The cfDNA was extracted from 2–5 mL plasma using the AVENIO cfDNA isolation kit (Roche Diagnostics), according to manufacturer’s instruction. In brief, the plasma sample was spun at 1800× g for 5 min to remove precipitates and debris, followed by proteinase K digestion. After the incubation with DNA Paraffin Binding Buffer (PBB) and isopropanol, the cfDNA was transferred to the High Pure Extender Assembly (HPEA) and the cfDNA was captured by the filter tube inside the HPEA. cfDNA was washed twice and eluted with DNA Elution Buffer, and then stored in −20 °C until use. cfDNA was quantified using a Qubit fluorometer 2.0 (Invitrogen Ltd., Life Technologies, USA) and a Qubit dsDNA HS Assay Kit (Invitrogen Ltd., Thermo Fisher Scientific), according to manufacturer’s instructions.
4
Liquid Biopsy Protocol for HCC
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We collected peripheral blood samples from each patient in Cell‐Free DNA Collection Tubes (Roche, Basel, Switzerland) before hepatectomy. cfDNA was extracted from 4 mL of plasma within 1 week preoperatively and postoperatively (at 1 week, 1 month, and 4 months, respectively) using the AVENIO cfDNA Isolation Kit (Roche). Fresh tumor specimens were gained from HCC patients based on a 7‐point baseline sample collection protocol during surgery [15 (link)]. DNA was extracted from the tissues using the All Prep DNA/RNA Mini Kit (Qiagen, Dusseldorf, Germany) according to the manufacturer's instructions.
5
Characterization of cell-free DNA
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The AVENIO cfDNA Isolation Kit (Roche Diagnostics) was used to isolate cfDNA from 2 mL of patient plasma, following the manufacturer’s protocol. The characteristic mononucleosomal molecular weight profile (160–200 bp) of the purified cfDNA was assessed using the Bioanalyzer 2100 High Sensitivity DNA Kit (Agilent Technologies). The cfDNA was quantified using the Qubit dsDNA High Sensitivity Kit (Thermo Fisher).
6
Plasma ctDNA Extraction and Quantification
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For plasma samples, 14 mL of peripheral blood was collected in EDTA-coated tubes before and 4 weeks after afatinib treatment, and upon disease progression. Plasma was separated by centrifugation at 1000 rpm for 15 min within 2 h of sample collection and stored at −80 °C until DNA extraction. Plasma ctDNA was purified using an AVENIO cfDNA isolation kit (Roche Diagnostics), and the quality and quantity of the DNA were verified using the NanoDrop 2000 device (Thermo Scientific) and PicoGreen dsDNA assay kit (Life Technologies) according to previous study11 (link). The extracted ctDNA was stored at −80 °C until the analysis.
7
Isolation and Purification of Cell-free DNA
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To separate whole blood into its constituent layers, samples were centrifuged in a free-bucket centrifuge at 1600 × g for 15 min. The top plasma layer was aspirated and aliquoted for long-term storage in vapour phase nitrogen tanks. The buffy coat layer was collected and further purified through erythrocyte lysis and phosphate-buffered saline washes, and resuspended in 1 mL of cell freezing media (50% RPM1, 40% FBS, 10% DMSO) for storage at −80 °C.
Cell-free DNA was extracted from 2 to 4 mL of plasma using either the column vacuum-based QIAGEN Circulating Nucleic Acid kit or the magnetic bead-based AVENIO cfDNA Isolation kit (Roche) following manufacturer protocols, and extracted ctDNA was eluted in 50–60 μL of AVE buffer or dH20, respectively. Even though a large majority of plasma DNA samples showed no genomic DNA contamination, routine QC was performed to ensure ctDNA sample quality. Using the Agilent 2100 Bioanalyzer and Agilent DNA High Sensitivity Kit, genomic contamination in ctDNA samples was identified by distinct high molecular weight fragments (>10,000 bp) in the electropherogram. Only samples with no genomic DNA contamination continued to amplicon panel sequencing.
Cell-free DNA was extracted from 2 to 4 mL of plasma using either the column vacuum-based QIAGEN Circulating Nucleic Acid kit or the magnetic bead-based AVENIO cfDNA Isolation kit (Roche) following manufacturer protocols, and extracted ctDNA was eluted in 50–60 μL of AVE buffer or dH20, respectively. Even though a large majority of plasma DNA samples showed no genomic DNA contamination, routine QC was performed to ensure ctDNA sample quality. Using the Agilent 2100 Bioanalyzer and Agilent DNA High Sensitivity Kit, genomic contamination in ctDNA samples was identified by distinct high molecular weight fragments (>10,000 bp) in the electropherogram. Only samples with no genomic DNA contamination continued to amplicon panel sequencing.
8
Plasma cfDNA Extraction and Quantification
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Molecular analyses were performed on patients complying clinical inclusion criteria and having adequate plasma DNA available.
cfDNA was extracted from 2 to 5 mL of plasma using the AVENIO cfDNA Isolation Kit (Roche Diagnostics Spa, Monza, Italia) and eluted into 60 μL of Elution Buffer, according to the manufacturer’s instructions. cfDNA was quantified using the QuBit dsDNA HS Assay kit with QuBit 3.0 fluorimeter (Thermo Fisher Scientific, San Jose, CA), and cfDNA quality was assessed by Agilent Bioanalyzer using a High Sensitivity kit (Agilent Technologies, Palo Alto, CA). The extracted cfDNA was stored at −20 °C until analysis.
cfDNA was extracted from 2 to 5 mL of plasma using the AVENIO cfDNA Isolation Kit (Roche Diagnostics Spa, Monza, Italia) and eluted into 60 μL of Elution Buffer, according to the manufacturer’s instructions. cfDNA was quantified using the QuBit dsDNA HS Assay kit with QuBit 3.0 fluorimeter (Thermo Fisher Scientific, San Jose, CA), and cfDNA quality was assessed by Agilent Bioanalyzer using a High Sensitivity kit (Agilent Technologies, Palo Alto, CA). The extracted cfDNA was stored at −20 °C until analysis.
9
Isolation and Characterization of cfDNA from Lung Cancer Patients
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cfDNA from lung cancer patients was isolated from 2 mL plasma using the AVENIO cfDNA Isolation Kit (Roche Diagnostics, Mannheim, Germany) following the manufacturer's instructions, and utilised for both capture-based targeted sequencing and shallow whole genome sequencing as detailed below. cfDNA from healthy control samples was isolated with the QIAamp MinElute ccfDNA Kit (Qiagen, Hilden, Germany). DNA was quantified with the Qubit dsDNA High Sensitivity Kit (ThermoFisher). Enrichment of the characteristic mononucleosomal fragment peak (160–200 bp) and absence of contaminating high molecular weight genomic DNA [28 ,29 (link)] were verified using the Bioanalyzer 2100 High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CA, USA).
10
Hybrid Capture Sequencing for cfDNA Profiling
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The AVENIO ctDNA expanded kit (Roche Diagnostics) is a hybridisation capture sequencing-based 77 genes pan-cancer assay (online supplemental table S1 ). AVENIO cfDNA Isolation Kit (Roche Diagnostics) was used to extract cfDNA from plasma according to the user’s manual. The extracted cfDNA was analysed by Agilent High Sensitivity DNA Analysis Kit (Agilent Technologies, California, USA) and Qubit dsDNA HS Assay Kit (ThermoFisher Scientific, California, USA) for quality control, and then used for library preparation with 10–50 ng cfDNA input. Prepared libraries were sequenced on the NextSeq 500 500/550 High Output Kit V2 (300 cycles) on Illumina NextSeq sequencing platform (Illumina, California, USA) and analysed by the AVENIO oncology analysis software V.2.0.0 (Roche Diagnostics) according to manufacturer’s instructions.24 25 (link)
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