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) <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 beta-globin (r=0.911, p<0.0001) and PicoGreen (r=0.915, p<0.0001) methods. CONCLUSIONS: Aside 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.
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 cancerpatients 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) <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 beta-globin (r=0.911, p<0.0001) and PicoGreen (r=0.915, p<0.0001) methods. CONCLUSIONS: Aside 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.
Authors: Lorena M Amaral; Valeria C Sandrim; Matthew E Kutcher; Frank T Spradley; Ricardo C Cavalli; Jose E Tanus-Santos; Ana C Palei Journal: Int J Mol Sci Date: 2021-01-08 Impact factor: 6.208