FIG. 3.

Linked RT-PCR and RT real-time PCR analyses of the NMB1966-to-NMB1958 region. (A) Genetic map of the NMB1966-to-NMB1958 region with the positions of the primer pairs (arrowheads) used in linked RT-PCR experiments. (B) Linked RT-PCR experiment. Total RNA from H44/76 grown to late logarithmic phase (OD₅₅₀ of 0.8) in complex GC medium was analyzed by RT-PCR using the following primer pairs: NMB1966-1/NMB1965-2 (lanes 2 and 3), NMB1965-1/NMB1964-r (lanes 4 and 5), NMB1964-f/NMB1963-2 (lanes 6 and 7), NMB1963-1/NMB1961-2 (lanes 8 and 9), NMB1961-1/NMB1960-2 (lanes 10 and 11), and NMB1960-1/NMB1958-2 (lanes 12 and 13). In lanes 3, 5, 7, 9, 11, and 13, Superscript RT was omitted as a control. The relative migration of DNA molecular weight ladders (lane 1) is indicated (bars on the left). (C) Semiquantitative analysis of the NMB1964-specific transcript in H44/76 and H44/76ΩgdhR by RT real-time PCR experiment. The RNA was extracted from meningococci grown to late logarithmic phase (OD₅₅₀ of 0.8) in GC medium. Results were normalized to 16S rRNA levels. Transcript levels in H44/76 are arbitrarily assumed to be equal to 100%. Values represent means from five independent experiments with triplicate samples using distinct cDNA preparations for each RNA sample ± standard deviations.