Optimization and comparison of genomic DNA extraction from whole blood collected in PAXgene blood RNA tube using automated platforms.
Author(s): You J, Shiomi T, Zappile P, Chiriboga L, Mendoza S, Moreira AL
Publication: Clin Chem Lab Med, 2025, Vol. , Page
PubMed ID: 41109964 PubMed Review Paper? No
Purpose of Paper
This paper used PAXgene-preserved blood to investigate the potential effects of blood thaw duration, freeze-thaw cycling, storage at 4°C, centrifugation speed and duration, washing the cell pellet, and DNA extraction method on the yield, integrity and size of genomic DNA. The effects of DNA extraction method on DNA amplificability were also investigated. The morphology of PAXgene-preserved blood cells was compared to cells from EDTA blood.
Conclusion of Paper
Blood cell smears from the pellet of PAXgene and EDTA blood both contained intact cells with identifiable nuclei, but the cells from PAXgene blood were smaller than EDTA blood. The DNA extracted directly from whole blood was brown in color and could not be quantified by a spectrophotometer or observed by gel electrophoresis. Washing the cell pellet prior to DNA extraction led to a slight, but nonsignificant, reduction in DNA yield, but clear bands >25 kb were observed regardless of the pellet washing protocol. DNA yields were not significantly affected by centrifugation speed (5,000 g, 10,0000 g, and 17,000 g) or duration (5 min versus 10 min), or thaw duration (recommended 2 h versus 0 or 1 h). DNA yield decreased progressively as the number of freeze-thaw cycles increased, with significantly lower yields from PAXgene blood that underwent five freeze-thaw cycles compared to PAXgene blood that underwent one cycle (P=0.0429). DNA levels were lower from PAXgene blood that was stored at 4°C for 2 or 4 weeks compared to blood stored for 0 weeks before centrifugation, and while the difference was not significant, the authors state PAXgene blood should not be stored at 4°C for ≥ 2 weeks.
There was a significant effect of extraction method on DNA yield (P=0.01), with much higher DNA yields when extraction was with the MAXwell RSC, DNeasy or QIAsymphony methods (48.52 ng/μL, 43.06 ng/μL and 42.73 ng/μL, respectively) than the Kingfisher system (1.36 ng/μL). However, the QIAsymphony method yielded DNA with the brightest band, the highest DNA integrity number (DIN, 9.40) and a main peak >60 kb. By comparison, the MAXwell RSC method yielded more DNA, but a less distinct DNA band, lower DIN (7.60), a much shorter main peak (17.24 kb) and less amplificability. The manual DNeasy Kit had comparable yields, amplificability and band intensity to the QIAsymphony method, but the DIN was slightly lower (8.53) and the main peak size slightly shorter (55.13 kb). The KingFisher method did not yield sufficient DNA for DIN measurement, but the main peak size was >60 kb and the DNA was amplifiable. Based on their results, the authors state that QIAsymphony SP is the best automated method, although the Maxwell RSC is also acceptable. When DNA was extracted from 96 specimens using the QIAsymphony SP in a 96-well format, just 12.5% of specimens yielded <1 μg.
Studies
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Study Purpose
This study used PAXgene-preserved blood to investigate the potential effects of blood thaw duration, freeze-thaw cycling, storage at 4°C, centrifugation speed and duration, washing the cell pellet, and DNA extraction method on the yield, integrity and size of genomic DNA. The effects of DNA extraction method on DNA amplificability were also investigated. The morphology of PAXgene-preserved blood cells was compared to cells from EDTA blood. Blood specimens were collected into PAXgene Blood RNA Tubes and K2EDTA Vacutainer Tubes from an unspecified number of patients (diagnosis not specified). PAXgene blood tubes were frozen at -80°C until extraction. Unless otherwise specified, PAXgene tubes were thawed at room temperature for 2 h, aliquoted, and the cells were pelleted by centrifugation at 17,000 g for 10 min and resuspended in phosphate-buffered saline; DNA was extracted using the DSP DNAMidi Kit using the QIAsymphony SP automated platform. To investigate the effects of pelleting and washing the blood cells, DNA was extracted from three PAXgene whole blood specimens and the washed or unwashed pellets obtained by centrifugation at 5000 g for 10 min. To investigate the effects of the centrifugation speed, cells from three PAXgene blood specimens were pelleted by centrifugation at 5,000 g, 10,0000 g, and 17,000 g for 10 min from matched aliquots. To investigate the effects of the centrifugation duration, cells were pelleted by centrifugation at 17,000 g for 5 or 10 min from matched aliquots of three thawed PAXgene blood specimens. To investigate the effects of post-thaw storage, matched aliquots of three thawed PAXgene blood specimens were stored at room temperature for 0, 1 or 2 h before centrifugation at 17,000 g for 10 min. To test the effects of freeze-thaw cycling, three PAXgene blood specimens were subjected to 1, 3 or 5 freeze-thaw cycles (details not provided). To investigate the effects of blood storage at 4°C, three PAXgene blood specimens were stored at 4°C for 0, 2 and 4 weeks before centrifugation. To investigate the effect of DNA extraction method, DNA was extracted from matched specimens using: 1) the DSP DNAMidi Kit and the QIAsymphony SP automated platform, 2) the Maxwell RSC Whole Blood DNA Kit and the Maxwell RSC automated platform, 3) the MagMAX DNA Multi-Sample Ultra 2.0 Kit and the KingFisher Apex automated platform, and 4) the manual DNeasy Blood & Tissue Kit. To test the efficiency of extraction, DNA was extracted from 96 PAXgene specimens using a 96-well plate format. DNA purity and concentration were evaluated by NanoDrop spectrophotometer. DNA integrity was assessed by gel electrophoresis and the Genomic DNA Screentape on a TapeStation 4200 Instrument. DNA integrity was verified by PCR amplification of a 295 bp region of DNA. Blood smears were H&E stained.
Summary of Findings:
Blood cell smears from the pellet of PAXgene and EDTA blood both contained intact cells with identifiable nuclei, but the cells from PAXgene blood were smaller than those from EDTA blood. The DNA extracted directly from whole blood was brown in color and could not be quantified by a spectrophotometer or observed by gel electrophoresis. Washing the cell pellet prior to DNA extraction led to a slight, but nonsignificant, reduction in DNA yield (2.03 ± 1.25 μg versus 3.09 ± 2.04 μg), but both had a clear band >25 kb. DNA yields were not significantly affected by centrifugation speed (6.27 ± 3.24 μg at 5,000 g, 6.90 ± 4.71 μg at 10,000 g, and 7.52 ± 4.39 μg at 17,000 g) or duration (3.87 ± 2.04 μg for 5 min and 4.85 ± 4.29 μg for 10 min). Further, there was no difference in DNA yield between specimens stored for the recommended 2 h post-thaw and those stored for 1 h or 0 h (3.55 ± 1.74 μg versus 3.24 ± 1.40 and 3.12 ± 1.57 μg, respectively). DNA yield decreased progressively as the number of freeze-thaw cycles increased, with significantly lower yields from PAXgene blood that was freeze-thawed 5 times compared to specimens that experienced one cycle (P=0.0429). DNA levels were lower when PAXgene blood was stored at 4°C for 2 or 4 weeks before centrifugation compared to specimens stored for 0 weeks, and while the difference was not significant, the authors state PAXgene blood should not be stored at 4°C for ≥ 2 weeks.
There was a significant effect of extraction method on DNA yield (P=0.01), with much higher DNA yields when extraction was with the MAXwell RSC, DNeasy or QIAsymphony methods (48.52 ng/μL, 43.06 ng/μL and 42.73 ng/μL, respectively) than the Kingfisher system (1.36 ng/μL). However, the QIAsymphony method yielded DNA with the brightest band, highest DIN (9.40) and a main peak >60 kb. By comparison, the MAXwell RSC method yielded more DNA, but a less distinct DNA band, lower DIN (7.60), a much shorter main peak (17.24 kb) and less amplificability. The manual DNeasy Kit produced comparable yields, amplificability and band intensity to the QIAsymphony method, but the DIN was slightly lower (8.53) and the main peak size slightly shorter (55.13 kb). The KingFisher method did not yield sufficient DNA for DIN measurement, but the main peak size was >60 kb, and the DNA was amplifiable. Based on their results, the authors state that QIAsymphony SP is the best automated method, although Maxwell RSC is also acceptable. When DNA was extracted from 96 specimens using the QIAsymphony SP in 96-well format, DNA yields ranged from 0.24 to 13.46 μg, with just 12.5% of specimens yielding <1 μg.
Biospecimens
Preservative Types
- Frozen
- PAXgene
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform DNA Automated electrophoresis/Bioanalyzer DNA Spectrophotometry Morphology H-and-E microscopy DNA PCR DNA Electrophoresis Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution PAXgene tube
EDTA tube
Storage Thaw duration 0 h
1 h
2 h (control)
Storage Storage duration 0 weeks at 4°C
2 weeks at 4°C
4 weeks at 4°C
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Multiple speeds compared
Multiple durations compared
Centrifuged
Not centrifuged
Storage Freeze/thaw cycling 1 cycle
3 cycles
5 cycles
Analyte Extraction and Purification Analyte purification The DSP DNAMidi Kit using the QIAsymphony SP automated platform
The Maxwell RSC Whole Blood DNA Kit using t the Maxwell RSC automated platform
The MagMAX DNA Multi-Sample Ultra 2.0 Kit using the KingFisher Apex automated platform
The manual DNeasy Blood & Tissue Kit
Biospecimen Preservation Type of fixation/preservation EDTA
PAXgene