title: DNA copy number concentration measured by digital and droplet digital quantitative PCR using certified reference materials.
abstract: The value assignment for properties of six certified reference materials (ERM-AD623a-f), each containing a plasmid DNA solution ranging from 1 million to 10 copies per μL, by using digital PCR (dPCR) with the BioMark™ HD System (Fluidigm) has been verified by applying droplet digital PCR (ddPCR) using the QX100 system (Bio-Rad). One of the critical factors in the measurement of copy number concentrations by digital PCR is the partition volume. Therefore, we determined the average droplet volume by optical microscopy, revealing an average droplet volume that is 8 % smaller than the droplet volume used as the defined parameter in the QuantaSoft software version 1.3.2.0 (Bio-Rad) to calculate the copy number concentration. This observation explains why copy number concentrations estimated with ddPCR and using an average droplet volume predefined in the QuantaSoft software were systematically lower than those measured by dPCR, creating a significant bias between the values obtained by these two techniques. The difference was not significant anymore when the measured droplet volume of 0.834 nL was used to estimate copy number concentrations. A new version of QuantaSoft software (version 1.6.6.0320), which has since been released with Bio-Rad's new QX200 systems and QX100 upgrades, uses a droplet volume of 0.85 nL as a defined parameter to calculate copy number concentration.
authors.affiliation: Institute for Reference Materials and Measurements, Joint Research Centre, European Commission, Retieseweg 111, 2440, Geel, Belgium, philippe.corbisier@ec.europa.eu.
authors.forename: Philippe
authors.initials: P
authors.lastname: Corbisier
authors.forename: Leonardo
authors.initials: L
authors.lastname: Pinheiro
authors.forename: Stéphane
authors.initials: S
authors.lastname: Mazoua
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Kortekaas
authors.forename: Pui Yan Jenny
authors.initials: PY
authors.lastname: Chung
authors.forename: Tsvetelina
authors.initials: T
authors.lastname: Gerganova
authors.forename: Gert
authors.initials: G
authors.lastname: Roebben
authors.forename: Hendrik
authors.initials: H
authors.lastname: Emons
authors.forename: Kerry
authors.initials: K
authors.lastname: Emslie
mesh_terms: D056915:DNA Copy Number Variations; D016133:Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards