Total RNA from bacterial cells was extracted using the TRIzol Reagent (Invitrogen) without DNA removing step (for RT-PCR and primer extension) or by using MasterPure™RNA Purification kit (Epicenter) with the removal of contaminated DNA (for microarray) [16, 21]. Immediately before harvesting, bacterial cultures were mixed with RNAprotect Bacteria Reagent (Qiagen) to minimize RNA degradation. RNA quality was monitored by agarose gel electrophoresis, and RNA quantity was determined using a spectrophotometer. Quantitative

RT-PCR Gene-specific primers were designed to produce a 150 to 200 bp amplicon for each gene. The contaminated DNA in RNA samples was removed using the Amibion’s DNA-free™Kit. cDNAs were generated using 5 μg of RNA and 3 μg of random hexamer primers. Using 3 independent cultures and RNA preparations, quantitative RT-PCR was performed in triplicate as described previously Geneticin molecular weight through the LightCycler system (Roche) together with the SYBR Green master mix [16, 21]. The PCR reaction mixture contained 2 μl of 10× PCRbuffer, 2 μl of this website 25 mmol/l MgCl2, 0.4 μl of 5 U/μl ExTaq DNA polymerase (Takala), 1 μl of 1:500 SYBR

Green I, 0.3 μl of each primer (10 μmol/l), 0.16 μl of 10 mmol/l dNTP, and 2 μl of cDNA templates, with the addition of H2O to arrive at a total volume of 20 μl. After pre-denaturation at 95°C for 3 min at a temperature transition rate of 20°C/s, PCR amplification was conducted at 45 cycles of denaturation at 95°C for 2 s at 20°C/s, annealing at 58°C for 4 s at 20°C/s and extension at 72°C for 8 s at 20°C/s, after which a single fluorescence measurement was taken at the end of the extension step. After amplification, a final melting curve was recorded by heating to 95°C, cooling to 65°C at 20°C/s, followed by a 60 s holding period at 65°C before heating slowly at 0.2°C/sec to 95°C. On the basis of the standard curves of

16 S rRNA expression, the relative mRNA level was determined by calculating Parvulin the threshold cycle (ΔCt) of each gene using the classic ΔCt method. Negative controls were performed using ‘cDNA’ generated without reverse transcriptase as templates. Reactions containing primer pairs without template were also included as blank controls. The 16 S rRNA gene was used as an internal control to normalize all the other genes [16]. The transcriptional AZD6244 datasheet variation between the WT and mutant strain was calculated for each gene. A mean ratio of two was taken as the cutoff of statistical significance. Primer extension assay For the primer extension assay [16, 21], about 10 μg of total RNA from each strain was annealed with 1 pmol of [γ-32P] end-labeled reverse primer. The extended reverse transcripts were generated as described in the protocol for Primer Extension System-AMV Reverse Transcriptase (Promega).