Specific real-time PCR vs. fluorescent dyes for serum free DNA quantification


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Abstract

BackgroundDetecting 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.MethodsSerum 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 β-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.ResultsThe 58 samples from healthy controls showed a reference limit of (95th percentile) <160 cyclophilin gene copies/mL. The 180 cancer samples displayed values ranging between 300 and 215,000 copies/mL. The cyclophilin method showed a high level of correlation with both the β-globin (r=0.911, p<0.0001) and PicoGreen (r=0.915, p<0.0001) methods.ConclusionsAside from the disadvantage that the QPCR assays can only be used in clinical biochemistry laboratories that possess QPCR apparatus, the use of direct PicoGreen quantification displays major advantages in a routine context: it is less time-consuming and is quite inexpensive, but is still correlated with QPCR.

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