Qiaamp circulating nucleic acid kit

Manufactured by Qiagen

Sourced in Germany, United States, Netherlands, United Kingdom, Italy, Spain, Japan, China, France

The QIAamp Circulating Nucleic Acid Kit is a laboratory equipment product designed for the purification of cell-free circulating nucleic acids (e.g., DNA, RNA) from various biofluid samples such as plasma, serum, or urine. The kit utilizes a spin column-based technology to efficiently extract and concentrate the target nucleic acids for further analysis.

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1 066 protocols using qiaamp circulating nucleic acid kit

Profiling Circulating Tumor DNA

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A total of 10 mL of peripheral blood was used as input material for library preparation. The supernatant following centrifugation for 10 min at 2000g at 4 °C was transferred to a new tube and centrifuged again at 16,000g at 4°C for 10 min. Subsequently, circulating free DNA was isolated from plasma using the QIAamp Circulating Nucleic Acid kit (Qiagen). Quality was verified by using the Qubit 2.0 Fluorimeter with the dsDNA HS assay kits (Life Technologies). A minimum of 50 ng of cfDNA is required for NGS library construction. Circulating free DNA was extracted using the QIAamp Circulating Nucleic Acid kit (Qiagen). Then subjected to end repair, phosphorylation and adaptor ligation. Fragments of size 200–400 bp were selected by AMPure beads (Agencourt AMPure XP Kit). Targeted DNA was captured, selected, and amplified. Quality of the fragments was assessed by using a bioanalyzer high‐sensitivity DNA assay. Indexed samples were sequenced in one lane on a Nextseq500 sequencer (Illumina, Inc.) with pair‐end reads. The mean coverage depth was 11,828×. Our assay captures 168 genes that are listed in the Gene list. The sequencing coverage and quality statistics, as well as the exact EGFR‐T790M mutation status are for each sample listed in the Table S1.

Chen Z., Liu L., Zhu F., Cai X., Zhao Y., Liang P., Ou L., Zhong R., Yu Z., Li C., Li J., Xiong S., Feng Y., Cheng B., Liang H., Xie Z., Liang W, & He J. (2022). Dynamic monitoring serum tumor markers to predict molecular features of EGFR‐mutated lung cancer during targeted therapy. Cancer Medicine, 11(16), 3115-3125.

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Extraction and Analysis of Cell-free DNA

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We isolated CSF cfDNA or total DNA from 1–10 mL CSF with the QIAamp Circulating Nucleic Acid Kit (Qiagen), and we isolated plasma cfDNA from 1–5 mL plasma with the QIAamp Circulating Nucleic Acid Kit. The blood cells separated from the blood samples were used for isolation of healthy DNA with the QIAamp DNA Mini kit (Qiagen). We isolated DNA from matched tumor tissue with the DNeasy Blood & Tissue kit (Qiagen). The size distribution of extracted DNA was measured by the Fragment Analyzer with the DNF-493 High Sensitivity Large Fragment Analysis Kit (Advanced Analytical).

Pan W., Gu W., Nagpal S., Gephart M.H, & Quake S.R. (2015). Brain Tumor Mutations Detected in Cerebral Spinal Fluid. Clinical chemistry, 61(3), 514-522.

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Automated cfDNA Isolation and Quantification

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cfDNA was isolated from plasma (QIAamp Circulating Nucleic Acid Kit, Qiagen) using a validated automated platform (QIASymphony, Quiagen).^{21 (link)} Samples were spiked with a known concentration of lambda DNA to assure adequate cfDNA recovery from plasma. cfDNA was measured using quantitative real-time PCR (Swift Biosciences, Ann Arbor, MI) with final concentrations expressed in ng/mL of plasma. cfDNA quality was confirmed by integrity score (QIAamp Circulating Nucleic Acid Kit, Qiagen) and high-sensitivity gel electrophoresis (Agilent). cfDNA samples with a high integrity score (≥ 0.70), suggesting the presence of longer DNA fragments characteristic of genomic origin, were excluded from analysis.

Brusca S.B., Elinoff J.M., Zou Y., Jang M.K., Kong H., Demirkale C.Y., Sun J., Seifuddin F., Pirooznia M., Valantine H.A., Tanba C., Chaturvedi A., Graninger G.M., Harper B., Chen L.Y., Cole J., Kanwar M., Benza R.L., Preston I.R., Agbor-Enoh S, & Solomon M.A. (2022). Plasma Cell-free DNA Predicts Survival and Maps Specific Sources of Injury in Pulmonary Arterial Hypertension. Circulation, 146(14), 1033-1045.

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Comparison of cfDNA Purification Kits

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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.

Nesic M., Bødker J.S., Terp S.K, & Dybkær K. (2021). Optimization of Preanalytical Variables for cfDNA Processing and Detection of ctDNA in Archival Plasma Samples. BioMed Research International, 2021, 5585148.

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Improved cfDNA Extraction from Plasma

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Example 2

Plasma samples were collected from patients and healthy individuals. ccfDNA was purified from the plasma sample using the present invention or QIAamp Circulating Nucleic Acid Kit (QIAGEN). As described in the previous examples, ATPS-paper stack and the control stack were immersed in the plasma samples, and wicked for approximately 2 minutes until the solution reached the top. The top was extracted and subjected to ethanol precipitation for Nanodrop analysis. The results were compared with the results obtained from QIAamp Circulating Nucleic Acid Kit (QIAGEN). Table 1 shows that the present invention can isolate cfDNA from plasma samples obtained from patients or healthy subjects at a higher yield than the commercial kit.

Table 1. Isolation and Concentration of ccfDNA from Serum Samples Obtained from Patients and Healthy Subjects Using the Present Method or QIAamp Circulating Nucleic Acid Kit

DNAIsolation methodYield (ng)

Patient samplePresent ATPS method184

QIAamp Circulating Nucleic Acid Kit89

Healthy controlPresent ATPS method27

QIAamp Circulating Nucleic Acid Kit5

US11366111B2. Method for accurate diagnosis of a disease targeting biomarkers in liquid biopsy (2022-06-21). Phase Scientific International, Ltd. [US]. Inventors: Yin To Chiu [HK].

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Genomic and cell-free DNA isolation

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Genomic DNA from MCF7 cells was isolated by proteinase K digestion at 65 °C for 30 min followed by purification using the QIAamp circulating nucleic acid kit (Qiagen, Venlo, The Netherlands). Genomic DNA from frozen tissue sections of colorectal liver metastases was isolated using the NucleoSpin Tissue kit according to the manufacturer’s guidelines. cfDNA was isolated from 2 ml of plasma using either the manual QIAamp circulating nucleic acid kit (Qiagen), or the semi-automated QiaSymphony DSP Circulating DNA Kit (Qiagen) and Maxwell® RSC ccfDNA Plasma Kit (Promega, Leiden, the Netherlands). DNA was eluted in elution buffers provided by the kits used [QIAamp kit: AVE buffer (RNase-free water with 0.04% NaN₃), QiaSymphony kit: ATE buffer (10 mM Tris–HCl pH 8.3, 0.1 mM EDTA, and 0.04% NaN3), Maxwell buffer (10 mM Tris, 0.1 mM EDTA, pH 9.0)] or RNAse-free water as specified.

Deger T., Boers R.G., de Weerd V., Angus L., van der Put M.M., Boers J.B., Azmani Z., van IJcken W.F., Grünhagen D.J., van Dessel L.F., Lolkema M.P., Verhoef C., Sleijfer S., Martens J.W., Gribnau J, & Wilting S.M. (2021). High-throughput and affordable genome-wide methylation profiling of circulating cell-free DNA by methylated DNA sequencing (MeD-seq) of LpnPI digested fragments. Clinical Epigenetics, 13, 196.

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Comparative Plasma DNA Extraction Kits

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For comparison of blood stabilisation and plasma volume the QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) was used. In addition, a comparison of three DNA extraction kits was performed on equal volumes of plasma (2 mL each) (Fig 1B). The following kits were used: PME free-circulating DNA Extraction Kit protocol (Analytik Jena, Jena, Germany) for 2–5 mL extractions using lysis solution GS/Binding solution VL system and DSP Virus/Pathogen Midi Kit performed on QIAsymphony (Qiagen) and the QIAamp Circulating Nucleic Acid Kit (QIAGEN). QIAsymphony extractions were performed at the Qiagen applications lab (Hilden). All plasma samples were processed according to the manufacturers’ protocols with the exception of the PME kit where an initial 20 min incubation instead of 10 min was performed and the plasma centrifugation steps were carried out at 3750 x g vs 4500 x g. All DNA was eluted in 80 μL within the range specified by the kit.

Sherwood J.L., Corcoran C., Brown H., Sharpe A.D., Musilova M, & Kohlmann A. (2016). Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA) from Patients with Non-Small Cell Lung Cancer (NSCLC). PLoS ONE, 11(2), e0150197.

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Metastatic Breast Cancer cfDNA Analysis

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Patients with metastatic breast cancer. CfDNA was isolated from 600 to 4000 µL plasma using the QIAGEN QIAamp Circulating Nucleic Acid kit (Qiagen) and eluted in 50 µL elution buffer. Depending on the amount of cfDNA 10 ng was used for NGS. Samples were concentrated using a speedvac concentrator if necessary. No ddPCR data were available for this cohort.
For all samples across cohorts, cfDNA concentrations were measured using the Quant‐iT dsDNA high‐sensitivity assay (Invitrogen, Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions, and the Qubit fluorometer (Invitrogen) was used as read out.

Bos M.K., Nasserinejad K., Jansen M.P., Angus L., Atmodimedjo P.N., de Jonge E., Dinjens W.N., van Schaik R.H., Del Re M., Dubbink H.J., Sleijfer S, & Martens J.W. (2020). Comparison of variant allele frequency and number of mutant molecules as units of measurement for circulating tumor DNA. Molecular Oncology, 15(1), 57-66.

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Blood Specimen Extraction and ctDNA Analysis

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We performed a series of blood specimen extractions from each patient at different time points. Peripheral blood drawn into 10-ml in ethylenediaminetetraacetic acid (EDTA) tubes were taken each time and processed immediately. For healthy volunteers, a single time point blood extraction was performed and plasma was extracted for ctDNA analysis. Blood plasma was obtained by centrifuging peripheral blood twice at 1000 g for 10 min at 4°C. The repeat centrifugation step was to remove any remaining contaminating cells from the supernatant from the first centrifugation. Cell-free DNA was purified using the Qiagen QIAamp Circulating Nucleic Acid kit (Qiagen Inc., USA) following the manufacturer’s instructions. Approximately 5 ml of plasma was processed from each sample and stored at −20°C prior to molecular analysis. Quantification of DNA was done using a Nanodrop 2000 device (Thermo Scientific, USA).

Wei Z., Wang W., Shu Z., Zhou X, & Zhang Y. (2017). Correlation Between Circulating Tumor DNA Levels and Response to Tyrosine Kinase Inhibitors (TKI) Treatment in Non-Small Cell Lung Cancer. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 23, 3627-3634.

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cfDNA Extraction and Library Prep

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DNA from blood was extracted using the QIAGEN QIAamp Circulating Nucleic Acid Kit (Qiagen). Genomic DNA from PBMCs was extracted using the DNeasy Blood and Tissue Kit (Qiagen). After extraction, genomic DNA was sheared to resemble cfDNA using an ultrasonicator (LE220, Covaris). Library preparation for TS, sWGS, and cfMeDIP was performed only for samples with >40 ng DNA yield. TS was excluded for samples with <40 ng DNA yield. Samples with <10 ng DNA yield were not processed.

Wong D., Luo P., Znassi N., Arteaga D.P., Gray D., Danesh A., Han M., Zhao E.Y., Pedersen S., Prokopec S., Sundaravadanam Y., Torti D., Marsh K., Keshavarzi S., Xu W., Krema H., Joshua A.M., Butler M.O, & Pugh T.J. (2023). Integrated, Longitudinal Analysis of Cell-free DNA in Uveal Melanoma. Cancer Research Communications, 3(2), 267-280.

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