FOR ISOLATION OF CFDNA FROM PLASMA FOR DOWNSTREAM APPLICATIONS
NextPrep-Mag™ cfDNA Automated Isolation Kit
- Ideal for extracting cfDNA from up to 5 mL human plasma and serum for cell-free DNA sequencing and other downstream applications
- Highly automated, mag-bead based format with robust yields
- Supported on the chemagic™ 360, chemagic™ MSM I, and chemagic™ Prime™ instruments
- Fast, 15 minutes hands-on-time and 1.25 hour turnaround for 24 samples using a 24-rod head or 96 samples using a 96-rod head
- Approximately 128 samples extractions possible per day
5 mL | 48 ISOLATIONS
5 mL | 240 ISOLATIONS
1.5 mL | 960 ISOLATIONS
1-3 mL | 16-50 ISOLATIONS
3-5 mL | 50 ISOLATIONS
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® Cell Free DNA-Seq Library Prep kit for Illumina® sequencing.
Automated cfDNA Isolation Protocol
The automated process allows for a significant decrease in total turnaround time and hands on time compared to current manual and partially automated solutions on-market.
POSITION PLASTICWARE ON DECK
ALIQUOT PLASMA TO PLATE, ADD PROTEINASE K & MAGNETIC BEADS
RUN SCRIPT FOR cfDNA ISOLATION (~1 HR)
REMOVE TUBES CONTAINING ELUTED cfDNA
NextPrep-Mag™ cfDNA Automated Isolation Kit Design Highlights
The kit offers a number of key advantages over other commercially available cfDNA extraction kits, including speed of cfDNA extraction while delivering robust yields and high concentration NGS libraries.
|Attributes||NextPrep-Mag™ cfDNA Automated Isolation Kit|
|Turnaround Time (24 samples)||1 hr 15 min|
|Hands-on Time||15 mins|
|Isolation Method||Magnetic beads|
|Temperature Consideration||Reagents shipped & all steps performed at room temperature|
|Automated Extraction Platforms||chemagic™ 360, chemagic™ Prime™, chemagic™ MSM I|
|Automated Prep Platform||Sciclone® and Zephyr® G3 NGS Workstation|
|Number of Heating Steps||None|
|Number of Steps on Ice||None|
|Carrier RNA Requirement||No|
|User Supplied Alcohol Requirement||No, included in kit|
|Plasma Input||5 mL on a 24-rod head system, 1.5 mL on a 96-rod head system (lower and higher input volume compatible)|
NextPrep-Mag™ cfDNA Automated Isolation Kit Performance
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.
Downstream sequencing application of cfDNA extracted using the NextPrep-Mag™ chemistry
Figure 3: Sequencing results from cfDNA extracted using NextPrep-Mag™ chemistry shows good concordance with expected mutation frequencies (A, B).
- 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
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.
- NextPrep-Mag™ cfDNA Isolation Kit: A High-throughput Solution for cfDNA Extraction from Plasma or Serum
The NextPrep-Mag™ cfDNA Automated Isolation Kit contains sufficient reagents for the indicated number of isolations from 1.5 mL or 4-5 mL of plasma/serum along with the accompanying disposable plastics for the chemagic™ 360 automation. The kit components are stable for at least 12 months from time of receipt, when properly stored.