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. 2013 Oct 4;8(10):e74946. doi: 10.1371/journal.pone.0074946

Figure 3. Primer competition in the multiplex pre-amplification PCR and Linearity/Dynamic range of the assay.

Figure 3

A) Known concentrations of Tbx21 (T), Gata3 (G), Rorγt (R), Foxp3 (F), Bcl-6 (B6), and ß-actin (B) DNA products (1, 1/10,1/100, 1/1000, 1/10000 and 1/100000 respectively), as well as, stat1, 3, 4 5A, 5B and stat6 (1, 1/10,1/100, 1/1000, 1/10000 and 1/100000 respectively) were mixed in the 1st round RT-PCR step. The different ratios of each product detected, reflect the initial dilutions of the different products, Cq were ∼2.8 cycles apart. The same concentration of 1/1000 was used for the all products; the Cq was ∼22.5 with a CV of 0.3%. C) ß-actin plasmid standards from 107 to 10 copies were used in the 2 step multiplex assay and a simplex real-time PCR assay. The r2 values for the simplex (triangle) and the multiplex (circle) reaction are r2 0.9904 and r2 0.9934 respectively. D) Bcl-6 plasmid standards from 107 to 10 copies were used in the 2 step multiplex assay and a simplex real-time PCR assay. The r2 values for the simplex (triangle) and the multiplex (circle) reaction are r2 0.9755 and r2 0.9872 respectively. All replicates were performed in triplicate.