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Evaluation of pre-analytical factors affecting plasma DNA analysis
Havell Markus1, Tania Contente-Cuomo1, Maria Farooq1
1Center for Noninvasive Diagnostics, Translational Genomics Research Institute, Phoenix, AZ, USA.
Scientific Reports
|May 11, 2018
Summary
A new multiplexed droplet digital PCR (ddPCR) assay helps assess circulating cell-free DNA (cfDNA) sample quality. This method reveals significant variations in cfDNA yield and fragment size due to different extraction kits, but not collection tubes.
Area of Science:
- Molecular Biology
- Genomics
- Biochemistry
Background:
- Pre-analytical factors critically influence circulating cell-free DNA (cfDNA) analysis.
- Robust methods for assessing cfDNA sample quality and pre-analytical processing effects are limited.
- Understanding these variations is crucial for reliable cfDNA biomarker studies.
Purpose of the Study:
- To develop a multiplexed droplet digital PCR (ddPCR) assay for assessing cfDNA sample quality.
- To evaluate the impact of different DNA extraction kits and blood collection tubes on cfDNA yield and fragment size.
- To identify pre-analytical variables affecting cfDNA integrity in clinical samples.
Main Methods:
- Developed a multiplexed ddPCR assay using 5 short and 4 long amplicons targeting single-copy genomic loci.
- Compared 7 cfDNA extraction kits for cfDNA yield and fragment size.
- Evaluated 3 blood collection protocols (EDTA tubes, Cell-free DNA Blood Collection Tubes) using plasma from 23 healthy volunteers.
Main Results:
- Significant variations in cfDNA yield and fragment size were observed across different cfDNA extraction kits.
- No significant differences in cfDNA yield, fragment size, or background noise were found between the tested blood collection protocols.
- cfDNA fragments were shorter in plasma processed immediately post-venipuncture compared to archived samples, indicating potential background DNA from lysed cells.
Conclusions:
- A multiplexed ddPCR assay is effective for assessing cfDNA sample quality before downstream molecular analyses.
- DNA extraction methods are a significant source of pre-analytical variation in cfDNA studies.
- Blood collection protocols evaluated did not introduce significant pre-analytical variation in cfDNA analysis.
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