The integrity, quality, and quantity of RNA were assessed using the Agilent Bioanalyser 2100. Microarray hybridizations selleck chemicals llc and data analysis The RNA labelling and hybridization were conducted by a commercial Affymetrix array service. An aliquot of 2 ug of total RNA was converted to double stranded cDNA with the one cycle cDNA Synthesis Kit, and then biotin tagged cRNA was produced with MessageAmp II aRNA Amplification Kit. The resulting bio tagged cRNA was fragmented to strands of 35 to 200 bases in length of the endogenous control gapdh gene, and then for a comparison between the expression of the gene in treated samples and in control samples. The delta Ct values of the gene in treated samples were subtracted by the delta Ct value of the gene in control samples.

The fold changes were cal culated by the formula of 2 delta delta Ct described by Livak Schmittgen. Data were means SD of tri plicate reactions for each gene transcript. Determining the effects of endotoxin from Salmonella typhimurium in chicken macrophages is an in vitro model to characterize the transcription profiles of one important cell type in the chickens immune response. Endotoxin is a complex lipopolysaccharide found in the outer cell membrane of Gram negative bacteria that is responsible for membrane organization and sta bility and differs from LPS in that it is a butanol water extract rather than a phenol water extract. Endotoxin used in the present study is between 10 and 20% protein and reproducible, hence its complexity better mimics the cell membrane in vivo.

Recognition of the lipid A and or the polysaccharide moiety of endotoxin by membrane receptors of monocytes induce a wide variety of cellular responses, including the synthesis of cytokines such as IL1B, TNF, IL6, IL8. Vertebrates have evolved an effective innate immune response to LPS containing bacteria over evolutionary time. Chickens are much more resistant than mammals to LPS induced septic shock and respond to LPS with the induction of IL1B, IL6, and IL18 mRNA. However, few studies have specifically examined the response to the more complex and more relevant immune stimulant, endotoxin, as a model for in vivo responses. Membrane bound receptors and also intracellular receptors such as NOD like Receptors play key roles in the recognition of pathogen associated molecular patterns to induce a host response. AV-951 Both receptor families contain a series of Leucine Rich Repeat modules in their ligand recognition domains. Although NLRs have been extensively studied in mammals, their regula tion in chicken is still to be described Macrophages play primary roles in both innate and adaptive immunity.