Quantitative Analysis Of Circulating DNA In Human Blood

BackgroundsCirculating DNA, also called cell-free DNA, is a kind of extracellular DNA present in the blood (plasma or serum), cerebrospinal fluid and synovial fluid. The development of sensitive techniques for the quantification of minute amounts of nucleic acids has provided powerful tools for the analysis of circulating DNA. While blood specimens could be easily accessible by a minimally invasive procedure, the finding of circulating cell-free DNA in the blood of healthy and diseased individuals has gained increasing attentions during the last years. Cell-free DNA concentrations increase in the circulating blood of patients with various diseases and may have clinical application potentials in diagnosis, prognosis and therapeutic effect. However, due to the variability of sample types, blood processing, storage of samples, DNA extraction procedures and techniques for quantification, the direct comparison of the numerous available publications is impossible and there are few large-scale studies concerning the normal reference range of circulating DNA levels in human blood.ObjectiveTo develop a novel duplex real-time PCR assay with internal control for the quantification of circulating DNA and to determine the normal reference range of circulating DNA levels in human blood.Materials and methodsAfter gene clone and multiple selections, a pMD18-T plasmid vector recombined with an artificial double-stranded DNA was quantified by spectrophotometric measurements and added as an internal control into plasma and serum samples of healthy adults before DNA extraction. Four DNA extraction protocols were evaluated for the recovery of recombinant plasmid DNA in plasma to choose an optimal protocol for the extraction of circulating DNA. A novel duplex real-time PCR assay was optimized for simultaneous amplifications of targetβ-actin gene and internal control recombinant plasmid DNA with three primers (using a common reverse primer) and two probes in the same reaction. The optimal concentrations of the Mg²⁺ and common reverse primer in the duplex PCR reaction were determined for best amplification efficiencies. The concentrations of circulating DNA was calculated by the concentrations of recombinant plasmid DNA. The sensitivity of this novel assay was determined by the quantification of plasma and serum samples with different volumes (200, 100, 50, 10, 2 and 1μl). The accuracy of this novel assay was determined by the quantification of human genomic DNA quantified by spectrophotometric measurements in plasma and serum. The plasma and serum DNA with differnet dilutions were quantified to evaluate the stability of this assay. The repeatability of this assay was also evaluated.The concentrations of circulating DNA in paired plasma and serum of 100 healthy adults were quantified and the associations with sex, age, total leukocyte counts, neutrophil counts and lymphocyte counts were analyzed. The normal reference ranges of human plasma and serum DNA levels were determined.ResultsA plasmid recombinated with artificial double-stranded DNA was verified by sequencing. There was no homology between recombinant plasmid and human genome. The chemagic DNA extraction kit yielded the highest recovery for recombinant plasmid DNA (71.4%) and the CV was 26%. The new developed duplex real-time PCR assay allowed simultaneous amplifications of targetβ-actin gene and intemal control recombinant plasmid DNA and two products of 91bp and 99bp could be well distinguished by two probes with two different fluoresceins JOE and FAM, respectively. The amplification efficiencies forβ-actin gene and recombinant plasmid DNA were 99.8% and 90.1%, while the concentrations of Mg²⁺ and reverse primer in PCR reaction were 6 mmol/L and 700 nmol/L.This assay allowed the quantification of plasma or serum samples with volume of as low as 2μl. The difference of quantitative results between this novel assay and spectrophotometric assay were below 20% and 30% for plasma and serum, respectively. There was no satistically significant difference among the quantitative results of plasma and serum DNA samples with diffemet dilutions (P＜0.05). The intra-assay and extra-assay CV were 11% and 17%, respectively.The concentrations of plasma and serum DNA both submitted to positive skewness distribution. The medians of the circulating DNA concentrations in paired plasma and serum of 100 healthy adults were 35 ng/ml (interquartile range, 24 ng/ml) and 283 ng/ml (interquartile range, 308 ng/ml), respectively. There was a statistically significant difference (P＜0.001) and obverse correlation (P＜0.001, r=0.52) between the paired plasma and serum samples. No statistically significant difference of plasma and serum DNA concentrations between male and female was found. There was also no association of plasma and serum DNA concentrations with age, total leukocyte counts, neutrophil counts and lymphocyte counts. At 95% confidence interval, the normal reference range of plasma and serum DNA concentrations were no more than 98 ng/ml and no more than 1.8μg/ml, respectively.ConclusionsA novel quantitative duplex real-time PCR assay with internal control for the quantification of circulating DNA in human blood has been established. It allows accurate measurements of circulating DNA in human blood with high sensitivity, specificity and repeatability even for plasma and serum samples with very small volume or DNA samples with different dilutions. This new developed assay has preliminary values for clinical applications. Plasma sample is more suitable for the quantification of cell-free circulating DNA than serum sample.