Title: DNA copy number concentration measured by digital and droplet digital quantitative PCR using certified reference materials
Authors: CORBISIER PhilippePINHEIRO LeonardoMAZOUA StephaneKORTEKAAS Anna MariaCHUNG PUI YAN JENNYGERGANOVA TSVETELINA IVANOVAROEBBEN GertEMONS HendrikEMSLIE K.
Citation: ANALYTICAL AND BIOANALYTICAL CHEMISTRY vol. 407 no. 7 p. 1831-1840
Publisher: SPRINGER HEIDELBERG
Publication Year: 2015
JRC N°: JRC92802
ISSN: 1618-2642
URI: http://publications.jrc.ec.europa.eu/repository/handle/JRC92802
DOI: 10.1007/s00216-015-8458-z
Type: Articles in periodicals and books
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 (BioRad). 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 defined parameter in the QuantaSoft software version 1.3.2.0 (BioRad) 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 BioRad’s new QX200 systems and QX100 upgrades, uses a droplet volume of 0.85 nL as a defined parameter to calculate copy number concentration.
JRC Directorate:Health, Consumers and Reference Materials

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