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cfDNA提取试剂盒 NextPrep-Mag™ cfDNA Automated Isolation Kit


Complete Solution for Isolation of Circulating DNA from Plasma for Downstream Applications

The NextPrep-Mag™ cfDNA Automated Isolation Kit is a complete solution for automated cell-free DNA extraction from human blood plasma or serum using the chemagic™ 360, chemagic™Prime™ or chemagic™MSM I instruments from PerkinElmer.


The challenges of extraction of cfDNA from plasma or serum are several, but are rooted in the fact that the circulating DNA is highly fragmented and present at low concentrations, especially in healthy individuals. The NextPrep-Mag™ cfDNA Automated Isolation Kit was developed to facilitate the workflow demands for high throughput labs while delivering optimal results.


The NextPrep-Mag™ cfDNA Automated Isolation Kit allows for the extraction of cell-free DNA from human blood plasma or serum  using the chemagic™ 360, chemagic™ Prime™ or chemagic™ MSM I instruments. The kit is all-inclusive and contains ready-to-use reagents and disposable plastics for 240 automated cfDNA isolations from up to 5 mL of starting plasma/serum. The cfDNA recovered can be used in a wide variety of downstream applications including next-gen sequencing and PCR analysis.


The cfDNA isolated with the NextPrep-Mag™ cfDNA kits have been verified to be compatible with both manual and automated NGS library construction on the Sciclone® G3 NGS workstation and the Zephyr® NGS workstation using the NEXTFLEX® Rapid DNA-Seq kit 2.0 for Illumina® sequencing.


货号产品名称规格
NOVA-3825-05NEXTprep-Mag™ cfDNA Isolation Kit (5 mL)240 isolations
NOVA-3825-06NEXTprep-Mag™ cfDNA Isolation Kit (5 mL)48 isolations
NOVA-3825-10NEXTprep-Mag™ cfDNA Isolation Kit (1.5 mL)960 isolations

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KIT CONTENTS

REAGENTS

cfDNA Lysis/Binding Solution

cfDNA Proteinase K

cfDNA Magnetic Beads

cfDNA Wash 1 Solution

cfDNA Wash 2 Solution

cfDNA Elution Solution (10 mM Tris/0.1 mM EDTA, pH 8)

chemagic™ 360 PLATFORM PLASTICS

Disposable tips

Rod-head specific plates, tubes, and elution tubes/plates

Please refer to respective protocol guides for details on plastics


REQUIRED MATERIALS NOT PROVIDED

Distilled deionized water used to dissolve Proteinase K

Vortex mixer

Pipettors

5 mL disposable pipets



The kit generated robust cfDNA recovery from both plasma and serum on the chemagic™ 360 and chemagic™ MSM I platforms as analyzed with electropherogram traces and Qubit® spectrophotometer.


NextPrep-Mag™ cfDNA Automated Isolation Kit 1.gif

Figure 1: Performance on the chemagic™ 360 Instrument. cfDNA was extracted from plasma (PL) or serum (SER) from 5 donors. (A) Yields were quantified electropherogram traces (for mononucleosome peak) and Qubit® spectrophotometer (for total DNA yield). (B) Overlay traces of triplicate 4 mL extractions.


NextPrep-Mag™ cfDNA Automated Isolation Kit 2.gif

Figure 2: The NextPrep-Mag™ cfDNA Automated Isolation Kit is robust on the chemagic™ MSM I instrument. Blood from different donors was collected in either (A) SST tubes or (B) Streck BCT tubes and extracted for serum. Serum was pooled from each collection method and 5 mLs were extracted using the automated protocol.


NextPrep-Mag™ cfDNA Automated Isolation Kit 3.gif

Figure 3: Sequencing results from cfDNA extracted using NextPrep-Mag™ chemistry shows good concordance with expected mutation frequencies (A, B).


Do we need special equipment to process large volumes of plasma?

No, the same kind of clinical centrifuges are used. For Input volumes less than or above 5 mL, slight modifications to the protocol may be required. Please inquire directly to Bioo.NGS@PerkinElmer.com..


What is the size distribution of cell free DNA recovered from human plasma?

The cell free DNA isolated from plasma of healthy donors, typically runs as a single small “hump” of ~ 150 bp when analyzed directly on an Agilent 2100 high sensitivity DNA chip. When the cell free DNA is converted into a library using the ligation-based format in the NEXTflex® Cell Free DNA-Seq Library Prep Kit, a greater range of sizes of library products is observed, from ~ 300 bp – 5,000 bp. The 5’ and 3’ adapters used to make the library add a total of ~150 bp. This indicates that cell free DNA fragments present at concentrations too low to be visualized directly are detectable after the amplification step used to create cell free DNA libraries.


Is it necessary to add carrier nucleic acid or glycogen when processing plasma to recover cell free DNA?

No. Addition of nucleic acid carrier compromises quantification and use of the cell free DNA as input for NGS libraries.


How many PCR cycles are needed to make cell free DNA libraries?

We typically use 9 – 12 PCR cycles for making cell free DNA libraries.

  • Sisson, B. A., Uvalic, J., Kelly, K., Selvam, P., Hesse, A. N., Ananda, G., . . . Reddi, H. V. (2019). Technical and Regulatory Considerations for Taking Liquid Biopsy to the Clinic: Validation of the JAX PlasmaMonitorTM Assay. Biomarker Insights,14, 117727191982654. doi:10.1177/1177271919826545

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