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Optimization of Sources of Circulating Cell-Free DNA Variability for Downstream Molecular Analysis
- Source :
- J Mol Diagn
- Publication Year :
- 2021
- Publisher :
- Elsevier BV, 2021.
-
Abstract
- Circulating cell-free DNA (ccfDNA) is used increasingly as a cancer biomarker for prognostication, as a correlate for tumor volume, or as input for downstream molecular analysis. Determining optimal blood processing and ccfDNA quantification are crucial for ccfDNA to serve as an accurate biomarker as it moves into the clinical realm. Whole blood was collected from 50 subjects, processed to plasma, and used immediately or frozen at -80°C. Plasma ccfDNA was extracted and concentration was assessed by real-time quantitative PCR (qPCR), fluorimetry, and droplet digital PCR (ddPCR). For the 24 plasma samples from metastatic pancreatic cancer patients, the variant allele fractions (VAF) of KRAS G12/13 pathogenic variants in circulating tumor DNA (ctDNA) were measured by ddPCR. Using a high-speed (16,000 × g) or slower-speed (4100 × g) second centrifugation step showed no difference in ccfDNA yield or ctDNA VAF. A two- versus three-spin centrifugation protocol also showed no difference in ccfDNA yield or ctDNA VAF. A higher yield was observed from fresh versus frozen plasma by qPCR and fluorimetry, whereas a higher yield was observed for frozen versus fresh plasma by ddPCR, however, no difference was observed in ctDNA VAF. Overall, our findings suggest factors to consider when implementing a ccfDNA extraction and quantification workflow in a research or clinical setting.
- Subjects :
- Real-Time Polymerase Chain Reaction
medicine.disease_cause
Circulating Tumor DNA
Pathology and Forensic Medicine
Cohort Studies
Biomarkers, Tumor
medicine
Humans
Digital polymerase chain reaction
Centrifugation
Neoplasm Metastasis
Alleles
Whole blood
Blood Specimen Collection
Chemistry
Regular Article
Molecular biology
Circulating Cell-Free DNA
Pancreatic Neoplasms
Real-time polymerase chain reaction
Molecular Diagnostic Techniques
Case-Control Studies
Mutation
Molecular Medicine
Biomarker (medicine)
Fresh frozen plasma
KRAS
Carcinoma, Pancreatic Ductal
Subjects
Details
- ISSN :
- 15251578
- Volume :
- 23
- Database :
- OpenAIRE
- Journal :
- The Journal of Molecular Diagnostics
- Accession number :
- edsair.doi.dedup.....ef407627d2b0842277092e087e0e4988
- Full Text :
- https://doi.org/10.1016/j.jmoldx.2021.08.007