title: Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material.
abstract: Digital polymerase chain reaction (dPCR) is a unique approach to measurement of the absolute copy number of target DNA without using external standards. However, the comparability of different dPCR platforms with respect to measurement of DNA copy number must be addressed before dPCR can be classified fundamentally as an absolute quantification technique. The comparability of four dPCR platforms with respect to accuracy and measurement uncertainty was investigated by using a certified plasmid reference material. Plasmid conformation was found to have a significant effect on droplet-based dPCR (QX100 and RainDrop) not shared with chip-based QuantStudio 12k or BioMark. The relative uncertainty of partition volume was determined to be 0.7%, 0.8%, 2.3% and 2.9% for BioMark, QX100, QuantStudio 12k and RainDrop, respectively. The measurements of the certified pNIM-001 plasmid made using the four dPCR platforms were corrected for partition volume and closely consistent with the certified value within the expended uncertainty. This demonstrated that the four dPCR platforms are of comparable effectiveness in quantifying DNA copy number. These findings provide an independent assessment of this method of determining DNA copy number when using different dPCR platforms and underline important factors that should be taken into consideration in the design of dPCR experiments.
authors: National Institute of Metrology, Beijing, 100029, P. R. China.
authors: Lianhua
authors: L
authors: Dong
authors: Hubei Institute of Measurement and Testing Technology, 430223, Wuhan, P. R. China.
authors: Ying
authors: Y
authors: Meng
authors: National Institute of Metrology, Beijing, 100029, P. R. China.
authors: Zhiwei
authors: Z
authors: Sui
authors: National Institute of Metrology, Beijing, 100029, P. R. China.
authors: Jing
authors: J
authors: Wang
authors: National Institute of Metrology, Beijing, 100029, P. R. China.
authors: Liqing
authors: L
authors: Wu
authors: National Institute of Metrology, Beijing, 100029, P. R. China.
authors: Boqiang
authors: B
authors: Fu
mesh_terms: D001483:Base Sequence; D002681:China; D056915:DNA Copy Number Variations / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics* / Q000592:standards*; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods / Q000592:standards*; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity