Important Procedure Notes when using the Monarch® Mag Cell-Free DNA (cfDNA) Extraction Kit (NEB #T4070)
Cell-free DNA detection
Cell-free DNA (cfDNA, ccfDNA, ctDNA) is an intrinsically challenging analyte to detect and characterize due to the fragmented nature, low concentration of DNA and high sample-to-sample variability. Our workflow enables efficient recovery of cell-free DNA by providing a reproducible extraction solution with low elution volume that minimizes loss, eliminating the need for additional concentration steps.
Downstream integration with NEB’s library prep and amplification platforms enables improved detection in end-to-end workflows.
Sample collection
Sample collection and preservation are crucial for cell-free DNA yield and recovery. Sample collection tubes and collection procedures should be carefully selected to stabilize cell-free nucleic acids and minimize contamination by cellular DNA. Tube manufacturer’s instructions for sample collection and storage should be followed. Our kit is compatible with several different collection tubes including standard anticoagulant tubes such as EDTA and sodium citrate, as well as preservative-containing tubes such as those offered by Zymo, Streck, Norgen and PAXgene.
DNA size recovered
The sample collection method used may impact the size range of recovered DNA and should be chosen appropriately for the intended application. The standard protocol of this kit enables efficient extraction of all sizes, including as low as 50 bp. If enhanced recovery of <100 bp fragments is required, a protocol modification is provided on the product web page for targeted recovery of ultrashort fragments (<100bp).
Sample types
This extraction kit has been evaluated for plasma and urine samples. Other suitable sample types include cerebrospinal fluid (CSF).
Sample handling and storage
During sample collection, follow the tube manufacturer’s instructions for best practices in sample draw and storage to preserve the cell-free DNA fraction and minimize cellular DNA release. If not using plasma for cfDNA extraction immediately upon blood processing, freeze the plasma at -20 °C or -80 °C.
Storing and handling of Monarch Mag Beads M2
Store Monarch Mag Beads M2 at 4°C after first use. For optimal performance, Monarch Mag Beads M2 should be equilibrated to room temperature before use. Importantly, the beads should be thoroughly vortexed right before dispensing into the sample.
Binding of cfDNA to beads
DNA binds to magnetic beads through interactions with the bead surface in the presence of the binding buffer (Monarch Buffer BX) and isopropanol. At this stage, the sample-binding mix needs to be mixed for 10 minutes to ensure efficient binding of cfDNA. A thermal mixer, shaker, or an end-over-end mixer is recommended.
If preferred, a binding mix of Monarch Buffer BX, Isopropanol, and Monarch Mag Beads M2 can be made for use within a day.
Bead drying
Following the final wash, beads should be dried thoroughly to remove residual ethanol. Optimally dried beads appear shiny and uniformly dark, while over-dried beads appear cracked or matte light brown. Avoid over-drying the beads. A drying time of 5 minutes is recommended with the tubes on the magnet and tube caps open.
Elution
At the elution step, DNA is released from the magnetic beads using nuclease-free water. Vigorous mixing on a thermal mixer or a vortexer is recommended to ensure complete release of DNA into the water. Elution volumes may be adjusted based on the desired DNA concentration. Use caution to not disturb the bead pellet when removing the eluate from the tube. It is normal to lose 1-2 µl when transferring the final elution into a new tube. For automated workflows, adjust the minimum elution volumes according to the system's specifications and requirements.
Volumes of reagents required based on extraction input volume
The scalability of this product allows for use of different sample input volumes with this kit.
Table 1. Volumes of reagents required based on extraction input volume. Choose an appropriately sized tube to accommodate the Total Sample Binding Mix.
|
|
For 1 ml sample |
For 2 ml sample |
For 3 ml sample |
For 4 ml sample |
|---|---|---|---|---|
|
Monarch StabiLyse DNA/RNA Buffer |
1 ml |
2 ml |
3 ml |
4 ml |
|
Proteinase K |
20 µl |
40 µl |
60 µl |
80 µl |
|
Monarch Buffer BX |
0.5 ml |
1 ml |
1.5 ml |
2 ml |
|
Isopropanol |
0.5 ml |
1 ml |
1.5 ml |
2 ml |
|
Monarch Mag Beads M2* |
30 µl |
60 µl |
90 µl |
120 µl |
|
Total Sample Binding Mix Volume |
3.05 ml |
6.1 ml |
9.15 ml |
12.2 ml |
|
Wash 1, Wash 2 and Wash 3 |
1 ml |
1 ml |
1 ml |
1 ml |
|
Elution |
15-100 µl |
20-100 µl |
40-100 µl |
50-100 µl |
*equilibrated to room temperature
Cell-free DNA Quantitation and Downstream Analysis
Due to the low concentration of cell-free DNA circulating in biofluids, UV-vis spectrophotometric methods are not recommended for quantification. The concentration of the extracted cell-free DNA can instead be measured using a sensitive fluorometric method such as Qubit™ 1X dsDNA High Sensitivity (HS) (Invitrogen). It is important to note that a Qubit method will also measure contaminating genomic DNA, if present in the sample. Therefore, a complementary assessment of fragment sizes using an automated electrophoresis such as Cell-free DNA ScreenTape Analysis (Agilent) is strongly recommended. Specific region settings on TapeStation software can be used enable the qualitative and quantitative measurement of extraction yields.
The extracted cell-free DNA is application-ready and can be used in sequencing or amplification-based detection. For sequencing-based analysis, NEBNext library prep solutions are recommended. NEBNext Ultra II DNA Library prep uses up to 50 µl input volume allowing for flexibility in upstream extraction elution volumes and the optimized library prep protocol for cfDNA ensures high conversion efficiency.
