title: Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation.
abstract: One of the benefits of Digital PCR (dPCR) is the potential for unparalleled precision enabling smaller fold change measurements. An example of an assessment that could benefit from such improved precision is the measurement of tumour-associated copy number variation (CNV) in the cell free DNA (cfDNA) fraction of patient blood plasma. To investigate the potential precision of dPCR and compare it with the established technique of quantitative PCR (qPCR), we used breast cancer cell lines to investigate HER2 gene amplification and modelled a range of different CNVs. We showed that, with equal experimental replication, dPCR could measure a smaller CNV than qPCR. As dPCR precision is directly dependent upon both the number of replicate measurements and the template concentration, we also developed a method to assist the design of dPCR experiments for measuring CNV. Using an existing model (based on Poisson and binomial distributions) to derive an expression for the variance inherent in dPCR, we produced a power calculation to define the experimental size required to reliably detect a given fold change at a given template concentration. This work will facilitate any future translation of dPCR to key diagnostic applications, such as cancer diagnostics and analysis of cfDNA.
authors.affiliation: LGC Limited, Queens Road, Teddington, Middlesex TW11 0LY, UK.
authors.forename: Alexandra S
authors.initials: AS
authors.lastname: Whale
authors.forename: Jim F
authors.initials: JF
authors.lastname: Huggett
authors.forename: Simon
authors.initials: S
authors.lastname: Cowen
authors.forename: Valerie
authors.initials: V
authors.lastname: Speirs
authors.forename: Jacqui
authors.initials: J
authors.lastname: Shaw
authors.forename: Stephen
authors.initials: S
authors.lastname: Ellison
authors.forename: Carole A
authors.initials: CA
authors.lastname: Foy
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Scott
mesh_terms: D045744:Cell Line, Tumor; D056915:DNA Copy Number Variations; D004273:DNA, Neoplasm / Q000737:chemistry*; D005260:Female; D005784:Gene Amplification; D018628:Gene Dosage; D018734:Genes, erbB-2; D006801:Humans; D046210:Microfluidic Analytical Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004273:DNA, Neoplasm