Isolation of cell-free DNA from plasma samples

Features

Magnet bead based isolation of cell-free DNA from 1–10 mL blood plasma

• Consistent cfDNA recovery from 1–10 mL plasma samples
• Efficient purification of fragmented DNA as small as 50 bp
• No PCR inhibition regardless of your preferred sample volume

Applications**

• Manual and automated isolation of circulating cell-free DNA from human plasma, derived from e.g., EDTA
blood draw tubes or Cell-Free DNA BCT® for “Liquid Biopsy” analysis**
• Typical downstream applications: NGS, qPCR, ddPCR

* Established on a KingFisher® Flex
** Kits to be used for research purposes only

Principle / Procedure

The NucleoMag® DNA Plasma kit is designed for the rapid manual and automated isolation of circulating cell-free DNA from human plasma, derived from e.g., EDTA blood draw tubes or Cell-Free DNA BCT®. The NucleoMag® DNA Plasma kit enables the recovery of fragmented DNA ≥ 50 bp with high efficiency due to a reversible adsorption of nucleic acids to paramagnetic beads under appropriate buffer conditions. Elution can be performed with as little as 50–200 μL elution buffer and the eluted DNA is ready to use for subsequent downstream applications like real-time PCR, digital droplet PCR, next generation sequencing, etc. The NucleoMag® DNA Plasma kit content is composed for cfDNA extractions of 2 mL plasma samples, but it can be scaled individually from 1 mL sample volume up to 10 mL

Sample lysis is achieved by incubation of a plasma with Liquid Proteinase K and Lysis Buffer MCF1 at 56 °C. For the adjustment of optimal binding conditions of nucleic acids and paramagnetic beads, Buffer MCF2 and the NucleoMag® P-Beads are added to the lysate. Three washing steps efficiently remove contaminating substances, such as PCR inhibitors, after magnetic separation. Subsequently residual ethanol from previous wash steps is removed by a drying step and highly pure DNA is finally eluted within 50–200 μL of a slightly alkaline Elution Buffer MCF5 of low ionic strength (5 mM Tris/HCl, pH 8.5) from the paramagnetic beads. Eluted DNA can be used directly for downstream applications. The NucleoMag® DNA Plasma kit can be used either manually or automated on standard liquid handling instruments or automated magnetic separators.

Application Data

The DNA yield strongly depends on the individual sample, but is typically in the range of 0.1 to 100 ng DNA per mL plasma. The NucleoMag® DNA Plasma kit enables the recovery of highly fragmented DNA ≥ 50 bp.

Reliable purification of cfDNA from different plasma volumes
The NucleoMag® DNA Plasma kit was used to isolate cfDNA from human EDTA plasma samples, ranging from 2–10 mL. Efficient purification of cfDNA is shown by the characteristic peak at approx. 170 bp using the Bioanalyzer™ 2100 system and the High Sensitivity DNA Kit from Agilent. The total yield was determined by PicoGreen™ quantification assay using the Quant-iT™ PicoGreen™ kit (Thermo Fisher Scientific) on a Synergy HT Multi-detection microplate reader (Biotek). The consistent correlation of cfDNA amount and sample volume demonstrates reliable performance independent on sample volume.

Competitive detection of low abundance cfDNA samples Total cfDNA from 2 mL human EDTA plasma derived from 4 challenging donor samples with low abundance cfDNA (< 10 ng cfDNA/mL Plasma) was purified. Isolation with the NucleoMag® DNA Plasma kit results in higher and more consistent total cfDNA yields with less fluctuations in comparison to the competitor T. The final total DNA recovery was quantified using the Qubit™ dsDNA High Sensitivity kit Thermo Fisher Scientific) on a Qubit™ fluorometer (Thermo Fisher Scientific).

Consistent cfDNA recovery regardless of plasma volumes The isolation of cfDNA from human EDTA plasma using the NucleoMag® DNA Plasma kit shows clear bands after measurement by capillary gel electrophoresis using the Agilent Bioanalyzer™ 2100 system with the High Sensitivity DNA kit. Even for low volumes such as 2 mL the detection of cfDNA is clearly visible.

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