Jul 14, 2025
gDNA extraction of nucleated blood from FTA elute cards
Peer-reviewed method
- Livia Gerber1,
- Sarah Whiteley2,
- Erin Hahn1,
- Clare Holleley1
- 1CSIRO;
- 2University of Canberra
- PLOS ONE Lab ProtocolsTech. support email: [email protected]
External link: https://doi.org/10.1371/journal.pone.0329019
Protocol Citation: Livia Gerber, Sarah Whiteley, Erin Hahn, Clare Holleley 2025. gDNA extraction of nucleated blood from FTA elute cards. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqnmq5gk5/v1
Manuscript citation:
Gerber L, Whiteley SL, Hahn EE, Holleley CE (2025) Stable preservation and recovery of methylation marks from FTA elute cards in species with nucleated red blood cells using a customized DNA extraction method. PLOS One 20(7). doi: 10.1371/journal.pone.0329019
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 16, 2024
Last Modified: July 14, 2025
Protocol Integer ID: 103466
Keywords: FTA card, Nucleated red blood cell, Archival DNA, FTA elute card, genomic DNA, DNA methylation, gdna extraction of nucleated blood, corresponding dna extraction protocol, dna extraction, cards suitable for most genomic library preparation, low molecular weight dna, gdna extraction, short dna fragment, protecting dna, high molecular weight dna, isolation of dna, sequencing technology, reliable storage of nucleic acid, molecular weight dna, dna, dna from degradation, majority of dna present, dna present, dna denaturation, nucleated blood, dna methylation, most genomic library preparation, sequencing application, fta card, nucleic acid, pcr amplification of short fragment, dna methylation mark, fta classic card, fta elute cards specialized, coated paper card, pcr, paper card, card matrix, card, using pcr amplification, specimen archive
Funders Acknowledgements:
CSIRO Future Science Platform
Grant ID: R-19568
Abstract
Specialized chemically-coated paper cards, such as Flinders Technology Associates (FTA) cards, provide simple and reliable storage of nucleic acids by protecting DNA from degradation. Owed to their simplicity, FTA cards are widely used in clinical testing, forensic science and specimen archives. Originally developed for PCR-based applications that only require short DNA fragments, FTA cards are now being explored as an avenue for whole-genome and epigenetic sequencing applications. FTA cards and their corresponding DNA extraction protocols have not kept pace with advances in sequencing technologies. Because the initial protocols developed for FTA cards were geared towards applications using PCR amplification of short fragments, they typically yield low molecular weight DNA. This issue is particularly pronounced for FTA elute cards where heat-based elution at 95°C leads to DNA denaturation and fragmentation. Isolation of DNA
from nucleated blood deposited onto FTA elute cards poses an additional challenge when compared to FTA classic cards, because hemoglobin is irreversibly bound to the card matrix, making the majority of DNA present in nucleated blood inaccessible. Here, we describe an easy, fast, and inexpensive protocol
to extract high molecular weight DNA (>10 kb) of nucleated blood stored on FTA elute cards suitable for most genomic library preparations including those that interrogate DNA methylation. Our protocol yields a 14-fold increase in yield compared to numerous alternatives. Using our protocol, we demonstrate that high
molecular weight DNA can still be extracted even after storage at ambient temperature for over a decade. Moreover, we show that DNA methylation marks are preserved on FTA elute cards, broadening the utility of FTA elute cards. This opens possibilities for (epi-)genomic studies using historical samples and enabling
specimen collection where access to chemicals or cryogenic storage is limited – reducing project costs and extending collection opportunities into remote areas.
Guidelines
The processing times for each step are indicative for a sample size of eight.
Materials
Equipment and consumables
FTA card punchers
2 ml safe-lock tubes and 1.5 or 2 ml tubes suitable for storage
Tips
High quality 5 mm stainless steel beads
Bunsen burner
Thermal shaker
Qubit or another reliable method for DNA quantification
Reagents
Ethanol for flaming
low TE buffer (10 mM Tris, 0.1 mM EDTA)
ddH2O
Proteinase K (20 mg/ml)
RNAse A (100 mg/ml)
Tris-Cl pH 8.5
Genomic DNA Clean & Concentrator-10 (Zymo D4010/4011)
Troubleshooting
Safety warnings
Ensure familiarisation with chemicals and choose appropriate PPE before commencing this protocol.
Ethics statement
University of Canberra Animal Ethics approval no. CEAE 17-08
CSIRO AEC 2024-12
Before start
- Label a 2 ml tube and a final storage tube (1.5 or 2 ml) for each sample.
- Sterilise the same number of 5 mm stainless steel beads as samples plus some spares.
- Set thermal shaker to 56°C.
Sample preparation
1h 10m
Punch two 3 mm punches from an FTA elute card into a 2 ml tube.
Note
Sterilise puncher (e.g. flaming) between samples to avoid cross-contamination.
30m
Wash punches twice with low TE and once with ddH2O. Each wash step is carried out in the 2 ml tube and consists of adding 500 μl of either TE (first two washes) or ddH2O (third wash), followed by vigorously vortexing for 5 s and subsequent removal of all 500 μl of wash solution but not punches.
Note
If more than eight samples are processed, carry out the washing step in batches of eight samples to avoid prolonged exposure of the punches to TE or ddH2O. Prolonged exposure negatively affects DNA yield.
40m
Proteinase K and RNA digest
18h 30m
Add 200 μl TE, 10 μl Proteinase K and a stainless bead to each sample.
Note
- The addition of the stainless steel bead is crucial. If no bead is added, DNA yield is significantly reduced.
- It is crucial to add a high quality stainless bead (e.g. Qiagen 5 mm stainless steel beads 69989). Low quality beads may rust, resulting in DNA degradation.
10m
Digest overnight at 56°C on a thermal shaker. Set shaker to 500 rpm.
16h
Remove samples from the thermal shaker and pulse-vortex each sample for 1 min. Add 10 μl Proteinase K to each sample.
15m
Place samples in the heat block and incubate 1-2 hours at 56°C, shaking at 800 rpm.
Expected result
The punches should no longer be intact after this step. If this is not the case, continue incubation and pulse-vortex the samples repeatedly.
Two completely lysed samples. The colour of the sample depends on the amount of blood on the punches. Samples with higher amounts appear more red compared to samples with smaller amounts of blood which appear grey.
1h 30m
Cool samples to room temperature. Add 1.5 μl RNAse A to each sample and digest for 30 minutes at 37°C. Shake at 500 rpm during RNA digest to break punches down even further.
35m
Extraction clean-up
1h 15m
Clean extract using Zymo genomic clean & concentrator according to manufacturer's instructions. We added 800 μl ChIp DNA binding buffer to the punch lysate and included a dry spin for 2 minutes after the second wash with wash buffer. We eluted the clean DNA in 25 μl heated (60°C) Tris-Cl (pH 8.5) to maximise DNA in our eluate.
Note
It may be possible to clean up the samples using SPRI beads. However, in a trial using SPRI beads DNA recovery was lower compared to the Zymo kit.
30m
Quantification and quality check
45m
Quantification:
We quantified the purified DNA extracts with a Qubit. We recommend the dsDNA BR over the dsDNA HS assay because some extracts may be out of range (too high) for the HS kit.
Purity assessment:
Purity of extracts can be assessed using a Nanodrop spectrophotometer or a similar device.
Fragment length estimation:
To evaluate fragment length, we used a TapeStation 4150.
Expected result
Using an elution volume of 25 μl, we obtained a mean DNA concentration of 101.25 ng/μl (lowest extract 17 ng/μl, highest 323 ng/μl) when measured using a Qubit 4 and the dsDNA BR assay.
Our extracts were of high purity (Nanodrop ratios: 260/230 mean = 2.3 ± 0.06; 260/280 mean = 1.9 ± 0.04).
From two 3mm punches containing nucleated blood, the overall yield is therefore approximately 2.5 μg with with an expected fragment size between 10 - 14 kb (measured on a TapeStation 4150 with the genomic DNA ScreenTape).
45m