A workflow for medium-throughput isolation of cfDNA from plasma samples using Apostle MiniMax™ on the KingFisher™ Technology
Brittany Niccum, PhD. Beckman Coulter Life Sciences. Indianapolis, INSummary
Cell-free DNA (cfDNA) is found in various bodily fluids. cfDNA has a characteristic size of approximately 175 bp. Due to its small size and low quantity the main challenge is to get enough cfDNA for sequencing. Subsequently, this results in a need to extract from larger volumes of bodily fluid, yet higher input volumes can be more difficult to manage for high-throughput sample processing.
This application note demonstrates the use of Apostle MiniMax™ High Efficiency cfDNA Isolation Kit, in conjunction with the KingFisher Duo Prime automated protein purification system. This potential solution that mitigates some of the challenges of processing large volume samples. Automating the chemistry can also reduce the risk of human error, reduce hands-on time and total time; therefore giving the user the ability to run more samples in a day.
Materials and Methods
Extraction of cfDNA from 1 mL and 2 mL plasma collected in EDTA tubes was semi-automated on the KingFisher™ Duo Prime using Apostle MiniMax™ High Efficiency cfDNA Isolation Kit (Standard Edition). Samples were stored at -80°C for >6 months and extractions took place during that time span. Prior to extraction, the plasma samples were thawed at room temperature. The plasma samples were first processed manually for lysis following the steps detailed in the protocol followed by cfDNA purification on the KinFisher Duo Prime. The second set of samples was extracted following a completely manual extraction following the Apostle MiniMax™ protocol.Manual processing
In this study, only one donor was used to assess the differences between manual and semi-automated cfDNA extraction. This donor was chosen due to previous work showing that this plasma sample had measurable quantities of cfDNA. Sample lysis buffer and proteinase K were added to the plasma sample. The sample was incubated at 60°C for 20 minutes. The tubes were cooled at the end of the incubation to room temperature. The cfDNA Lysis/Binding Solution and Magnetic Nanoparticles were added to the sample followed by a 10 minute incubation at room temperature while shaking. The plate was then moved to the KingFisher™ Duo Prime for further extraction.Automated processing
The parameters for mix times and speeds and the collect times were optimized for the magnetic beads on the KingFisher™ Duo Prime. The optimized parameters and reagent volumes for a 1 mL sample of plasma collected in an EDTA tube can be seen in Table 1 and for a 2 mL sample of plasma collected in an EDTA tube can be seen in Table 2.| DNA
Purification
Step |
Plate |
Plate Row |
Reagent |
Volume
(μL) |
Automation parameters |
|
| Mixing time/ Mixing Speed | Collect Count/ time [s] | |||||
| Bind |
1 |
A |
Lysate | 1140 | 1 min/ Medium |
8/ 30 sec |
| cfDNA Lysis/Binding Solution | 1250 | |||||
| Magnetic NanoParticles | 15 | |||||
| Wash 1 | 1 | B | cfDNA Wash Solution | 1000 | 1 min/ Medium | 3/ 30 sec |
| Wash 1 | 1 | C | cfDNA Wash Solution | 1000 | 1 min/ Medium | 3/ 30 sec |
| NA | 1 | D | KingFisher™ Duo 6-Tip Comb | NA | ||
| Wash 2 |
2 |
A |
cfDNA 2nd Wash Solution | 200 | 1 min/ Medium |
5/ 30 sec |
| 100% Ethanol | 800 | |||||
| Wash 2 |
2 |
B |
cfDNA 2nd Wash Solution | 200 | 1 min/ Medium |
5/ 30 sec |
| 100% Ethanol | 800 | |||||
| NA | 2 | C | NA | NA | ||
| Elution | 2 | D | cfDNA Elution Solution | 20 | 1 min/ Medium | 3/ 30 sec |
Table 1. KingFisher™ Duo Prime parameters for cfDNA extraction from a 1 mL plasma sample.
| DNA
Purification
Step |
Plate |
Plate Row |
Reagent |
Volume
(μL) |
Automation parameters |
|
| Mixing time/ Mixing Speed | Collect Count/ time [s] | |||||
| Bind |
1 |
A |
Lysate | 2280 | 1 min/ Medium |
8/ 30 sec |
| cfDNA Lysis/Binding Solution | 2500 | |||||
| Magnetic NanoParticles | 30 | |||||
| Wash 1 | 1 | B | cfDNA Wash Solution | 1000 | 1 min/ Medium | 3/ 30 sec |
| Wash 1 | 1 | C | cfDNA Wash Solution | 1000 | 1 min/ Medium | 3/ 30 sec |
| NA | 1 | D | KingFisher™ Duo 6-Tip Comb | NA | ||
| Wash 2 |
2 |
A |
cfDNA 2nd Wash Solution | 200 | 1 min/ Medium |
5/ 30 sec |
| 100% Ethanol | 800 | |||||
| Wash 2 |
2 |
B |
cfDNA 2nd Wash Solution | 200 | 1 min/ Medium |
5/ 30 sec |
| 100% Ethanol | 800 | |||||
| NA | 2 | C | NA | NA | ||
| Elution | 2 | D | cfDNA Elution Solution | 40 | 1 min/ Medium | 3/ 30 sec |
Table 2. KingFisher™ Duo Prime parameters for cfDNA extraction from a 2 mL plasma sample.
Results
The manual extractions were compared to the extractions done on the KingFisher™ Duo Prime. The total yields were compared and presented in Figure 1. The yield of DNA was estimated by using Quant-it™ dsDNA Assay Kit. The yield varied for the manual and automated extraction for the two different input volumes due to experimental variation. The coefficient of variation was calculated for the 4 different groups (Table3). For both of the plasma volumes the coefficient of variation for the automated methods was less than for the manual method indicating that there is less variability in relation to the average yield when processing on the KingFisher™ Duo Prime.
Due to the volume limitations on the KingFisher™ Duo Prime the largest input sample volume is 2 mL. If a larger volume is desired the sample volumes can be split between multiple wells and the
elution can be combined.
Figure 1. The cfDNA extracted using the Apostle MiniMax™ on KingFisher™ Duo Prime and by Manual extraction. The error bars are representative of the standard deviation of three technical replicates.
| Volume of Plasma | KingFisher™ Duo Prime | Manual |
| 1 mL | 4.4% | 5.0% |
| 2 mL | 9.6% | 10.0% |
Table 3. The coefficient of variation for the 4 different conditions
Conclusions
Using a semi-automated platform it is demonstrated to reduce hands-on-time significantly without sacrificing cfDNA yield (table 3). Automation can also help reduce the risk for human errors as there are less human intervention steps. The throughput of the KingFisher™ Duo Prime is only 6 samples per run due to the large volumes required to extract sufficient cfDNA from plasma. The overall protocol does not require significant time, and at max capacity in an 8 hour day, 36 samples can be processed with only 30 minutes of total hands-on-time.
A supplemental protocol is also available for processing a 2 mL sample with the KingFisher™ Flex.
| Throughput per run | Hands-on Time | Walk away Time | Total Time | |
| Manual | 24 | 45 minutes | 30 minutes | 75 minutes |
| Semi-automated KingFisher™ Duo | 6 | 5 minutes | 70 minutes | 75 minutes |
Table 4. The throughput and time for manual and semi-automated cfDNA extraction
Beckman Coulter makes no warranties of any kind whatsoever express or implied, with respect to this protocol, including but not limited to warranties of fitness for a particular purpose or merchantability or that the protocol is non-infringing. All warranties are expressly disclaimed. Your use of the method is solely at your own risk, without recourse to Beckman Coulter. Not intended or validated for use in the diagnosis of disease or other conditions. This protocol is for demonstration only, and is not validated by Beckman Coulter.
KingFisher, KingFisher Duo Prime, and Quant-iT™ dsDNA Assay (PN: Q33120) are trademarks of Thermo Fisher Scientific, Inc .
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