Fig 1.
Correlation between qPCR and duplex ddPCR measurements.
qPCR and ddPCR data were represented as normalized expression level of FKBP5 and ratio of FKBP5 and TBP (copies/μl) respectively. Each dot represents a sample of trials.
Fig 2.
Simplex and duplex ddPCR assays.
1D droplet plot of simplex ddPCR assay to measure FKBP5 and TBP gene expression (A). 1D droplet plot of duplex ddPCR assay combining the FKBP5 and TBP assays into a single reaction and employing primer concentrations of 100 nM (B), and 50 nM and 150 nM respectively (C). 2D droplet plot of the FKBP5/TBP duplex assay (D) based on different primer concentrations. Blue, green and black dots represent FKBP5, TBP amplicons and negative droplets respectively. Orange dots correspond to droplets containing both gene amplicons.
Fig 3.
Normalized expression level of target gene.
(A) FKBP5 gene expression was detected in six samples of the bovine thymus by means of simplex and duplex ddPCR assay using EvaGreen dye. Error bars indicate the standard deviation of the measure triplicate. (B) Correlation analysis between simplex and duplex ddPCR measurements. The results are presented as the ratio of copies/μl of FKBP5 and TBP.
Table 1.
Intra-assay (test precision) and inter-assay variation (test variability) of duplex ddPCR using six samples of cDNA as start template.
Fig 4.
ROC curve for FKBP5 down-regulation GC treated veal calves (A) and young bulls (B) versus untreated control animals (trial 1 and trials 2 and 3 respectively).