Unknown,Transcriptomics,Genomics,Proteomics

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QRT-PCR of ex vivo Salmonella enterica in TLR4 minus/plus macrophage background

ABSTRACT: A major virulence factor in S. enterica is the pathogenicity island Spi2. The goal of the experiment was to determine the impact of the toll-like receptor 4 (TLR-4) on expression of Spi2 in S. enterica wild type (WT) and triple mutant strain (TMS) with deletions in SPI-1 TTSS, SPI-2 TTSS, flagella. The stain 14028 was used for the experiment. Cells were grown in a TLR-4 wildtype (plus) or deleted (minus) macrophage background. Total RNA was extracted and processed by qRT-PCR. Two strains (WT and TMS) grown under 2 conditions (TLR-4 wildtype (plus) or TLR-4 deleted (minus)) - total 4 samples were analyzed in the study. All PCRs were done in quadruplicates, located on the same PCR plate. For each quadruplicates the median value was calculated and used as a data point for further normalization. A set of controls was selected for all data sets using the following criteria: the data points for the controls should have Cp <40 and be present in all the samples.

ORGANISM(S): Salmonella enterica subsp. enterica serovar Typhimurium str. LT2

SUBMITTER: John Braisted

PROVIDER: E-GEOD-26804 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The innate immune system is vital to rapidly responding to pathogens and Toll-like receptors (TLRs) are a critical component of this response. Nanovesicular exosomes play a role in immunity, but to date their exact contribution to the dissemination of the TLR response is unknown. To understand the effect of exosomal cargo released from locally stimulated cells on distal cell expression, we collected exosomes from local ovarian adenocarcinoma (HEY) cells that were either unstimulated (control-exosomes), stimulated with pIC (pIC-exosomes), or lipopolysaccharide (LPS-exosomes) for 48 hours. The three groups of exosomes were added to naÃ¯ve (distal) cells and the gene expression profiles were compared between local TLR stimulation (for 6 hours) and distal stimulation mediated by exosomes at the 48-hour time point The goal of the study was to delineate the differential effector function of TLR-exosomes based on the innate immune activation state (control-, LPS-, pIC-stimulation) of the cell of origin in vitro. We used microarrays to understand the changes in gene expression in 2 samples of local hey cells either unstimulated(control) or stimulated with poly(I:C) or LPS each. We also profiled 2 samples of distal naÃ¯ve cells that were exposed to either control, LPS or poly(I:C) exosomes from local cells. Here we show that exosomes from TLR stimulated cells (TLR-exosomes) can largely recapitulate TLR activation in distal cells in vitro. We can abrogate the action-at-a-distance signaling of exosomes by UV irradiation, demonstrating that RNA is crucial for their effector function. This work definitively establishes the differential effector function for TLR-exosomes in communicating the activation state of the cell of origin. HEY cells were either unstimulated (Local control) or stimulated with LPS or poly (I:C) for 6 hours and the RNA from these cells was profiled using a microarray. Exosomes were collected from local cells that were unstimulated or stimulated for 48 hours and added to distal (naive) HEY cells that were exposed to control exosomes, LPS exosomes or pIC exosomes and then the RNA was collected for analysis by microarray. mRNA expression was captured on Affymetrix U133 Plus 2 chips. mRNA microarray data were analyzed using the Expression Console software (Affymetrix) and Bioconductor tools written in the R statistical programming language. Pre-processing of raw signal intensities and normalization was performed using GCRMA (R). Linear modelling of the transformed data was determined by using Limma in R with the Benjamini and Hochberg correction. Differentially expressed probesets were identified using a threshold 5% FDR correction and a fold change â¥ 1.4 was applied

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QRT-PCR of ex vivo Salmonella enterica in TLR4 minus/plus macrophage background

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