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.

Graphical Abstract

Monolayer of droplets generated by the droplet generator and observed under an optical microscope

Fig. 1

Discrimination of the positive and negative droplets using the BCR-ABL ddPCR assay on the ERM-AD623 reference material series. The positive droplets are represented in green, whereas the negative droplets are coloured in grey

Fig. 2

Comparison of both digital PCR platforms using the certified reference material ERM-AD623 as test material. Horizontal error bars represent the expanded uncertainty associated to the reference material value assigned by dPCR, whereas the vertical bars represent the standard deviation based on eight ddPCR replicates

Fig. 3

Typical image taken of a monolayer of droplet generated by the droplet generator and observed under an optical microscope (A) as well as the image treated by the ImageJ software (v1.47q) to distinguish the droplets from each other and to differentiate the in-between droplet areas from the droplet areas (B)

Fig. 4

Influence of the value used for average droplet volume in Eq. (1) on the copy number concentration (displayed with their standard deviation) determined by ddPCR. (a) With using a volume of 0.91 nL used in the QuantaSoft version 1.3.2.0, (b) with using an average volume of 0.834 nL measured in this study and (c) with using an average volume of 0.833 nL measured by NMIA. The continuous line represents the certified value assigned by dPCR and the dotted line displays the 95 % lower confidence interval

Fig. 5

Relationship between the average amplitude of the fluorescence in negative (A) and positive (B) droplets in dependence on the droplet size. Each diamond represents the average droplet volume and the average fluorescence amplitude of droplets generated from a single well in a droplet generator cartridge