FIG. 4.
Analysis of plsX gene expression by quantitative RT-PCR. RT-PCRs were performed as described for Fig. 3, except that different masses of competitive DNA were added to a given mass of RT-PCR mixture. (A) Quantitative RT-PCR with primers 1 and 7. Different masses (15 × 10−5, 12.5 × 10−5, 10 × 10−5, 7.5 × 10−5, 5 × 10−5, 2.5 × 10−5, or 2.25 × 10−5 ng) of competitive DNA amplified from pYZ64 with primers 1 and 7 were added to the RT-PCR mixtures in lanes 1 through 8, respectively. (B) Quantitative RT-PCR with primers 6 and 5. Competitive DNA (7.5 × 10−5, 6.5 × 10−5, 4.5 × 10−5, 3.5 × 10−5, 2.5 × 10−5, 2 × 10−5, or 1 × 10−5 ng) was added to lanes 1 through 8, respectively. The fluorescence intensities of the bands on the agarose gel in each lane were quantified with densitometry following ethidium bromide staining. The ratio of the intensity of the PCR product of competitive DNA to that of the RT-PCR product was calculated for each reaction, and these ratios were plotted as a function of the competitive DNA concentration. (C and D) Plots of data from panels A and B, respectively.