Real-time quantitative PCR analysis of mice lung tissue (2). Mus musculus

Recombinant-murine S100A8 and the corresponding Cys42 to Ala42 mutant were used at 10 mg/50 ml HBSS administered onto the nares of BALB/C mice. To assess direct effects, mice were sacrificed 1, 4, 6, 12 or 20 h post-inhalation of S100A8 or 12 and 20 h post-inhalation of Ala42S100A8. For comparison with Dex inhalation (used at 10 mg/50 ml HBSS), lungs were harvested 6 and 12 h post administration. Because S100A8 is reported to initiate proinflammatory responses by ligating TLR4 and/or RAGE, a quantitative PCR array was developed to analyse 63 genes, selected to reflect potential acute inflammatory changes induced by ligation of these receptors. Overall design: Expression of inflammatory genes was evaluated with the RT-qPCR array. Relative quantities of mRNA in duplicate samples were obtained using the LightCycler® 480 Software 1.5 and the Efficiency-Method.