NextPrep-Mag™ cfDNA Automated Isolation Kit Performance
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.
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.
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.
Figure 3: cfDNA extraction yields and profiles. cfDNA was extracted in triplicate on 2 separate days from 1 mL of plasma in 2 K2EDTA and 2 Streck® BCT® tubes using NextPrep-Mag™ chemistry. Yields were quantified using the Thermo Fisher® Scientific Qubit® HS DNA assay (left). DNA profiles were observed using the PerkinElmer NGS3K assay on a LabChip® GXII Touch™ instrument (right).
Downstream sequencing application of cfDNA extracted using the NextPrep-Mag™ chemistry
Figure 4: Sequencing results from cfDNA extracted using NextPrep-Mag™ chemistry shows good concordance with expected mutation frequencies (A, B).
NextPrep-Mag™ cfDNA Automated Isolation Kit Citations
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
- 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
- 5 mL disposable pipets
chemagic™ 360 PLATFORM REQUIREMENTS
- Please refer to respective protocol guides for details for each rod-head configuration
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 [email protected]
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.
The NextPrep-Mag™ cfDNA Automated Isolation 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 on a 24-rod head system, and 960 isolations from up to 1.5 mL on a 96-rod head system. The kit components are stable for at least 12 months from time of receipt, when properly stored.