1. Specific real-time PCR vs. fluorescent dyes for serum free DNA quantification
- Author
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Stéphane Moutereau, Jerôme Lemant, Véronique Barbu, Mory Sacko, Michel Vaubourdolle, Mihelaiti Chiminqgi, Marc Conti, Pascal Pernet, and Sylvain Loric
- Subjects
Adult ,Clinical Biochemistry ,Context (language use) ,Biology ,Polymerase Chain Reaction ,Cyclophilins ,Cyclophilin A ,chemistry.chemical_compound ,Neoplasms ,Humans ,Gene ,Cyclophilin ,Aged ,Fluorescent Dyes ,Biochemistry (medical) ,DNA ,General Medicine ,Middle Aged ,Fluorescence ,Molecular biology ,Globins ,Standard curve ,Real-time polymerase chain reaction ,chemistry ,Case-Control Studies ,Reagent Kits, Diagnostic - Abstract
BACKGROUND Detecting and quantifying circulating free DNA in patient serum has become a major challenge. New methods using conventional or automated DNA amplification have been developed. As quantitative real-time PCR (QPCR) remains expensive and requires dedicated automated instrumentation, we questioned whether simple quantification using fluorescent dyes is efficient for determination of free DNA levels in serum. METHODS Serum samples from 180 cancer patients and 58 healthy volunteers were used for DNA quantification according to three methods: (i) using an exonic part of the beta-globin gene as the amplifying target; (ii) amplifying a 105-bp intron 1 part of the housekeeping cyclophilin A gene, both referring to specific standard curves; and (iii) using a PicoGreen DNA quantification kit without amplification. RESULTS The 58 samples from healthy controls showed a reference limit of (95th percentile)
- Published
- 2007
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