BackgroundDuplex real-time PCR assays have been widely used to determine amounts and concentrations of free circulating DNA in human blood plasma samples. Circulatory plasma DNA is highly fragmented and hence a PCR-based determination of DNA concentration may be affected by the limited availability of full-length targets in the DNA sample. This leads to inaccuracies when counting PCR target copy numbers as whole genome equivalents.Methodology/Principal FindingsA model system was designed allowing for assessment of bias in a duplex real-time PCR research assay. We collected blood plasma samples from male donors in pools of 6 to 8 individuals. Circulatory plasma DNA was extracted and separated by agarose gel electrophoresis. Separated DNA was recovered from the gel in discrete size fractions and analyzed with different duplex real-time PCR Taqman assays detecting a Y chromosome-specific target and an autosomal target. The real-time PCR research assays used differed significantly in their ability to determine the correct copy number ratio of 0.5 between Y chromosome and autosome targets in DNA of male origin. Longer PCR targets did not amplify quantitatively in circulatory DNA, due to limited presence of full-length target sequence in the sample.ConclusionsPCR targets of the same small size are preferred over longer targets when comparing fractional circulatory DNA concentrations by real-time PCR. As an example, a DYS14/18S duplex real-time PCR research assay is presented that correctly measures the fractional concentration of male DNA in a male/female mixture of circulatory, fragmented DNA.

Fragmented DNA and RNA circulate as cell-free nucleic acids in plasma, serum, urine and other body fluids. Access to these molecules for analysis may allow for detection of certain disease states based on a blood sample. In this study the extraction efficiency of a large volume nucleic acid extraction kit for circulating and viral nucleic acids was assayed with different downstream qPCR assays. For circulating DNA in plasma the yield was 7-to 9-fold higher with the large volume kit compared to the QIAamp DNA Blood Mini protocol with improved recovery of short DNA fragments. The microRNA extraction protocol of the large volume kit was found to deliver significantly increased microRNA yields ( Ct from 3 to 4) compared to the kit's standard protocol. For viral nucleic acids, 95% detection rates were: HBV DNA 0.25 IU/ml and HIV-1 RNA 14.2 IU/ml. The results demonstrate that the QIAamp Circulating Nucleic Acid Kit can serve as a sample preparation solution for processing up to 5 ml cell-free body fluid, it allows for small elution volumes, and can extract and concentrate circulating nucleic acids, including microRNA, and viral nucleic acids up to 250 fold.