Melt-curve analysis was included to identify nonspecific products. All RNA samples were tested for DNA contamination using a one-step RT-PCR kit with SYBR Green (Bio-Rad Laboratories) lacking reverse transcriptase. For RNA analysis, the program of the iCycler was Deforolimus price as follows: RT reaction for 10 min at 50 °C, followed by 5 min at 95 °C. The PCR was carried out in 45 cycles consisting of denaturation for 10 s at 95 °C and elongation for 30 s at 60 °C. A final denaturation step of 1 min at 95 °C and a
final elongation step for 1 min at 55 °C were also conducted. For DNA analysis, the program was as indicated but excluded the initial cDNA synthesis step (10 min at 50 °C). To determine the half-lives of different types of RNA, we assumed that the amount y of a specific RNA at time t was given by an exponential function, where y0 and T represent the initial amount of RNA and the half-life, respectively. For each of the graphs, we determined which values of the constants y0 and T minimized the least square error. The values of T obtained by this procedure are given in Table 2. Statistical analysis was performed using Student’s t-test (two-tailed distribution, two-sample
equal variance) when indicated in the figure legends. Mean relative amounts of each target mRNA, normalized buy 17-AAG to individual control RNA, were added together and divided with the corresponding number of control RNAs (four control RNAs). During a C. pneumoniae infection, the amount of DNA and the number of bacteria increase between 14 and 26 h p.i., but not before that time (Ouellette et al., 2006, Fig. 1). Also, the microorganisms differentiate from metabolically inactive EBs to metabolically active RBs before 14 h p.i. (Wolf et al., 2000). Flucloronide On the contrary, addition of the growth inhibitor INP0010 abolished C. pneumoniae proliferation and the amount of DNA increased only slightly between 2 and 26 h p.i. (Fig. 1, Bailey et al., 2007). To further analyze the mechanism of INP0010, it was of interest to measure gene expression in INP0010-treated and untreated C. pneumoniae during the transition phase (14 h p.i.).
We chose to investigate several genes coding for components of the virulence-associated type 3 secretion system (T3SS), as well as the gene groEL_1, which encodes the housekeeping chaperone GroEL (Table 3). Expression of these mRNAs was correlated with different control RNAs [16S rRNA, rpoA, rpoD, and gyrA (Goellner et al., 2006; Bailey et al., 2007; Fink et al., 2007)]. Data obtained in previous experiments had indicated that treatment with INP0010 reduced the transcription of some T3SS genes when 16S rRNA was used as an internal control (Bailey et al., 2007). Therefore, to examine the effect of INP0010 on T3SS gene expression when using different internal expression controls, we allowed C. pneumoniae to infect HEp-2 cells in the presence or the absence of INP0010 for 14 h.