Specific real-time PCR vs. fluorescent dyes for serum free DNA quantification.
Author(s): Chiminqgi M, Moutereau S, Pernet P, Conti M, Barbu V, Lemant J, Sacko M, Vaubourdolle M, Loric S
Publication: Clin Chem Lab Med, 2007, Vol. 45, Page 993-5
PubMed ID: 17867987 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to compare cell-free DNA quantification methods for serum, and to determine whether diagnosis affects serum DNA levels.
Conclusion of Paper
DNA concentrations, determined by real-time PCR of cyclophilin A, were very strongly correlated with those determined by real-time PCR of β-globin or by PicoGreen. DNA concentrations, determined by real-time PCR of β-globin, were strongly correlated with those determined by PicoGreen. Serum from cancer patients had significantly higher DNA concentrations than serum from healthy individuals.
Studies
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Study Purpose
The purpose of this study was to compare DNA quantification methods in serum specimens by real-time PCR amplification of β-globin or cyclophilin A, or the fluorometric PicoGreen assay. The effect of DNA diagnosis on serum DNA levels was also investigated. DNA was extracted from serum collected from 87 rectocolic cancer, 93 prostate cancer and 58 healthy patients using the QIAamp blood kit. Extracted DNA was stored at -20ºC in polypropylene tubes until quantification.
Summary of Findings:
The calculated number of copies of DNA per mL were very strongly correlated when quantified by real-time PCR amplification of β-globin and cyclophilin A (r=0.911, p<0.0001). Similarly, the number of copies of DNA per mL determined by PicoGreen and real-time PCR of β-globin were strongly correlated (r=0.81, p<0.0001), and the number of copies of DNA per mL determined by PicoGreen and real-time PCR of cyclophilin A were very strongly correlated (r=0.915, p<0.0001). Serum from cancer patients had significantly higher levels of DNA than serum from healthy individuals (p<0.0001).
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Fluorometry DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Real-time qPCR Specific Targeted nucleic acid β-globin exon
Cyclophilin A intron
Preaquisition Diagnosis/ patient condition Cancer
Healthy
Real-time qPCR Specific Technology platform PicoGreen