Figure 2

Semi-quantitative analysis of priA -specific transcripts in intracellular meningococci. (A) RT-PCR slot blot analysis of the priA-specific transcripts. Total RNAs were extracted from meningococci (strain B1940) after 7 h-infection of HeLa cells (intracellular), or control bacteria grown for 7 h in culture medium (control) and were used to generate gene-specific ³²P-labeled cDNA probes as detailed in the Methods section. The ³²P-labeled cDNA probes were hybridized to different amounts (4, 20 and 100 ng) of denatured NMB0551 (priA)-specific DNA fragments. For the 16S rRNA gene-specific fragment, two-fold serial dilutions (from 0.05 to 50 ng) were used. (B) Densitometry analysis of the RT-PCR slot blot with 20 ng of priA-specific DNA fragments. The relative transcript levels of priA in intracellular meningococci are arbitrarily assumed equal to 100%. Values represent means from five independent experiments, each with triplicate samples, using RNA preparations from distinct infection assays. Bars indicate standard deviations. (C) Semi-quantitative analysis of the priA-specific transcripts by RT real-time PCR experiment. The RNAs were extracted from intracellular or control meningococci as described above. Results were normalized to 16S rRNA levels. Transcript levels of priA in intracellular meningococci were arbitrarily given a value of 100%. Data are shown as mean ± standard deviation from five independent experiments, each with triplicate samples, using RNA preparations from distinct infection assays. The Student's T-test was used for statistical analysis. Statistically significant differences between values from intracellular and control bacteria (asterisks) are declared at a p value < 0.05.