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Nucleosnap cfdna kit

Manufactured by Macherey-Nagel
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Sourced in Germany

The NucleoSnap cfDNA kit is a product designed for the rapid and efficient isolation of cell-free DNA (cfDNA) from various sample types. The kit utilizes a simple and straightforward procedure to extract cfDNA, which can be used for downstream applications such as PCR, sequencing, or other molecular analysis techniques.

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Lab products found in correlation

Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions) Dneasy blood tissue kit, by Qiagen (1 mentions) Qiaamp dna ffpe tissue kit, by Qiagen (1 mentions) Nucleospin mirna plasma kit, by Macherey-Nagel (1 mentions) Nf h2o, by Thermo Fisher Scientific (1 mentions) Qiaamp dna investigator kit, by Qiagen (1 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions) High sensitivity d1000 assay, by Agilent Technologies (1 mentions) Accel ngs 2s hyb dna library kit, by Integrated DNA Technologies (1 mentions) Picogreen dsdna assay, by Thermo Fisher Scientific (1 mentions) Novaseq 6000, by Illumina (1 mentions) Tapestation system, by Agilent Technologies (1 mentions) Qubit 3 fluorometer, by Thermo Fisher Scientific (1 mentions)

5 protocols using nucleosnap cfdna kit

1

Plasma Separation and DNA Extraction Protocol

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Up to 40 mL (range 6–40 mL) of blood were collected in 5 × 6-mL or 4 × 10-mL, purple-capped EDTA tubes. Consistent with published recommendations, blood plasma was isolated within 6 hours of collection by first spinning whole blood at 1000g for 10 min, separating the top plasma layer into 1 mL aliquots, then spinning those aliquots at 15,000g for 10 min, transferring the supernatant to cryovials, and storing at −80°C.77 (link) DNA extraction of tumor tissue from FFPE was carried out using QIAamp DNA FFPE Tissue Kit (QIAGEN). DNA was extracted from plasma using the NucleoSnap cfDNA kit (Macherey-Nagel) and from buffy coat using the DNeasy Blood & Tissue Kit (QIAGEN). DNA isolated from both FFPE samples and buffy coat were fragmented by sonication to 150 bp using a Covaris E220 prior to library preparation.
Johnson B.E., Creason A.L., Stommel J.M., Keck J.M., Parmar S., Betts C.B., Blucher A., Boniface C., Bucher E., Burlingame E., Camp T., Chin K., Eng J., Estabrook J., Feiler H.S., Heskett M.B., Hu Z., Kolodzie A., Kong B.L., Labrie M., Lee J., Leyshock P., Mitri S., Patterson J., Riesterer J.L., Sivagnanam S., Somers J., Sudar D., Thibault G., Weeder B.R., Zheng C., Nan X., Thompson R.F., Heiser L.M., Spellman P.T., Thomas G., Demir E., Chang Y.H., Coussens L.M., Guimaraes A.R., Corless C., Goecks J., Bergan R., Mitri Z., Mills G.B, & Gray J.W. (2022). An omic and multidimensional spatial atlas from serial biopsies of an evolving metastatic breast cancer. Cell Reports Medicine, 3(2), 100525.
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2

Comparative Analysis of cfDNA and cfRNA Extraction Kits

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Due to NucleoSnap cfDNA kit’s (Macherey-Nagel, Düren, Germany) superior performance in the first comparative experiments, we directly compared NucleoSnap to the NucleoSpin miRNA Plasma kit (Macherey-Nagel, Düren, Germany) concerning cfDNA yield and purity. Moreover, we evaluated cfRNA yields from both kits. For NucleoSpin, all samples were centrifuged at 4500× g before the start of the protocol. Skipping the optional DNA digestion step allowed for the parallel extraction of cfDNA and cfRNA. The final elution volume was 50 µL nuclease-free water (nf-H2O; Thermo Fisher Scientific, Waltham, MA, USA) after 5 min incubation on the membrane. Co-isolated cfNA was stored at −80 °C until further analysis.
Maass K.K., Schad P.S., Finster A.M., Puranachot P., Rosing F., Wedig T., Schwarz N., Stumpf N., Pfister S.M, & Pajtler K.W. (2021). From Sampling to Sequencing: A Liquid Biopsy Pre-Analytic Workflow to Maximize Multi-Layer Genomic Information from a Single Tube. Cancers, 13(12), 3002.
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3

Isolation and Purification of Circulating Cell-Free DNA

Cited 1 time
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A free and open access protocol for the procedure is available online (10.17504/protocols.io.81wgb6z9olpk/v1). Briefly, hemolymphatic LBs were collected from the abductor muscle as described [22 (link)]. The hemolymph was clarified by centrifugation at 1200 × g for 3 min, and the supernatant was frozen (−20 °C) until use. To isolate and purify ccfDNA, samples (1.5–2.0 mL) were thawed and processed using the NucleoSnap cfDNA kit (Macherey-Nagel, Bethlehen, PA) according to the manufacturer’s instructions. The ccfDNA was stored at −80 °C until further analysis. In some experiments, ccfDNA was extracted and purified using the QIAamp DNA Investigator Kit (QIAGEN, Toronto, ON, Canada). Purified DNA was quantified by standard PicoGreen assay. The fragment distribution of the extracted ccfDNA was analyzed by capillary electrophoresis with an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA) using a High Sensitivity DNA kit. The assay was performed according to the manufacturer’s instructions using 1 μL of ccfDNA sample.
Ferchiou S., Caza F., de Boissel P.G., Villemur R, & St-Pierre Y. (2022). Applying the concept of liquid biopsy to monitor the microbial biodiversity of marine coastal ecosystems. ISME Communications, 2, 61.
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4

Liquid Biopsy cfDNA Extraction and NGS

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Supernatants from pre-processed CSF samples were thawed and processed using the NucleoSnap cfDNA kit (Macherey-Nagel) according to the manufacturer’s instruction (Figure 1B). Elution was performed with 50 µl of 5 mM Tris-HCL. Extracted cfDNA was quantified by qPCR using primers for the ALU sequences referenced against serially diluted human genomic DNA (1 pg/µl – 10 ng/µl, 10-fold dilutions, Promega) (Iqbal et al., 2015 (link)). Size distribution of cfDNA was assessed using the TapeStation System and High Sensitivity D1000 Assay (Agilent). For MRD detection using lcWGS, up to 2 ng or 40 µl of cfDNA was used without fragmentation as input for library preparation with the Accel-NGS 2S Hyb DNA Library Kit (Swift Biosciences). Dephosphorylation, end repair, and ligation of single-indexing adapters were performed according to protocol while library amplification was guided by a pilot quantitative PCR run. No template controls were included in each experiment. Libraries were quantified by the PicoGreen dsDNA Assay (Invitrogen), pooled, and subjected to 100bp paired-end sequencing on the NovaSeq 6000 instrument (Illumina) targeting for 3x coverage.
Liu A.P., Smith K.S., Kumar R., Paul L., Bihannic L., Lin T., Maass K.K., Pajtler K.W., Chintagumpala M., Su J.M., Bouffet E., Fisher M.J., Gururangan S., Cohn R., Hassall T., Hansford J.R., Klimo P J.r., Boop F.A., Stewart C.F., Harreld J.H., Merchant T.E., Tatevossian R.G., Neale G., Lear M., Klco J.M., Orr B.A., Ellison D.W., Gilbertson R.J., Onar-Thomas A., Gajjar A., Robinson G.W, & Northcott P.A. (2021). Serial assessment of measurable residual disease in medulloblastoma liquid biopsies. Cancer cell, 39(11), 1519-1530.e4.
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5

Circulating Cell-Free DNA Isolation

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Plasma was thawn and cfDNA was isolated from up to 2 ml using the NucleoSnap cfDNA kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s procotol. cfDNA was quantified using the Qubit dsDNA HS Assay kit and the Qubit Fluorometer 3.0 (Thermo Fisher Scientific). DNA size distribution was assessed on the Bioanalyzer instrument using the DNA High Sensitivity Kit (Bioanalyzer, CA, USA).
Gahlawat A.W., Witte T., Sinn P, & Schott S. (2023). Circulating cf-miRNA as a more appropriate surrogate liquid biopsy marker than cfDNA for ovarian cancer. Scientific Reports, 13, 5503.
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