70). Aliquots for RNA analysis were taken from each bacterial culture and placed in RNAProtect. An additional aliquot was taken from each culture for a cell culture invasion assay. All experiments were performed four separate times. Salmonella invasion assays The aliquots taken following the 30 minute incubation with and without tetracycline were centrifuged at 16,000 x g for 2 minutes, and the pellets were re-suspended in fresh LB broth to remove the antibiotic. Invasion assays were performed with technical replicates for each biological replicate using a gentamicin protection assay in HEp-2 cells at a multiplicity
of infection of ~40 as previously described . Percent invasion GSK690693 in vitro was calculated by dividing CFU/ml recovered by CFU/ml added. The significance of the differences in invasion were determined by a one-way repeated measures ANOVA with Dunnett’s post-test to assess pair-wise differences between the no-antibiotic control and the other sample conditions using GraphPad Prism 5. P values less than 0.05 were considered significant. Each isolate had a different invasion rate without tetracycline, therefore check details invasion
at 1, 4, and 16 μg/ml tetracycline was normalized to the control for each isolate at each growth phase for graphical representation of the fold change; the complete pre-normalized invasion data can be found in Additional file 1. Real-Time PCR assays RNA was isolated using the RNeasy Mini Kit (QIAGEN, Germantown, MD), and genomic DNA was removed using the Turbo DNase DNA-free Demeclocycline kit (Ambion, Austin, TX) according to the directions from the manufacturers. Total RNA was quantitated
on a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE). Reverse transcription was carried out using the Applied Biosystems High capacity cDNA reverse transcription kit on total RNA using random primers (Life Technologies, Grand Island, NY), and technical replicates were performed for each biological replicate. Real-Time PCR was performed in a Bio-Rad CFX96 Real-Time PCR Detection System (BioRad Laboratories, Hercules, CA) using the SYBR Green Master Mix (Applied Biosystems, Foster City, CA). Primer sets were used to evaluate the 16S rRNA, hilA, prgH, invF, tetA, tetB, tetC, tetD, and tetG transcripts (Table 2). For control assays, reverse transcriptase was not added to AZD1480 parallel mixtures for each sample. Amplification was performed using the following cycle conditions: 95°C for 10 min; 40 cycles of 95°C for 15 s, 55°C for 30 s, 72°C for 30 s; melting curve analysis from 65°C to 95°C. Raw data was analyzed using LinRegPCR software, and amplification efficiencies and cycle threhhold (CT) values were determined using a Window of Linearity for each primer set .