Broader applications for the quantitative single-cell multiplex RT-PCR. Aliquots of cDNA from human small intestine were tested as described in Figure 2. (A,B) Efficiency of PCR amplification for different gene products. Aliquots were amplified separately for each gene and each type of PCR. (A) Quadruplicate amplification slopes for Ccl-5, Gzma, first PCR (solid lines) and second PCR (dash lines). Amplification of all genes gave the same results (B) Slope values from the quadruplicates were assessed on the exponential phase; efficiency and significance of variation were performed as described in Figure 2. Results show mean ± SD of PCR efficiencies of the first (upper panel) and second (bottom panel) PCRs. All primer combinations had the same efficiency. We also found no significant difference in the variance between the first and second PCR amplifications (ANOVA, P > 0.05). Data are from one of three independent experiments. (C,D) Competition: Aliquots of cDNA from human small intestine were amplified separately for each gene, or in multiplex in the first PCR round. Quadruplicates of these reactions were further amplified in a second real-time PCR. (C) Quadruplicates of amplification for Ifn-γ, Gzmb genes amplified in multiplex (solid line) or alone (dashed line). Comparison of CT values obtained for each different gene amplified in multiplex (black bars) or separately (gray bars). No significant differences were observed between the two amplification conditions for each different gene (t-test, P > 0.1).
