Mice were exposed to large tidal volume (17 ml/kg, 6 hrs or 35 ml/kg, 2 hrs) using a small animal mechanical ventilator with non-mechanical ventilated, spontaneous breathing mice used as a control (n=2). Sample_age: 7-8 weeks old Sample_hybridization: total RNA was converted to first-strand cDNA using a hybrid reverse transcription primer consisting of oligo-dT and T7 RNA polymerase promoter sequences. The single-stranded cDNA was then converted to double-stranded cDNA. Complementary DNA corresponding to 5-10 µg of total RNA was used in a cRNA amplification step using T7 RNA polymerase and two biotinylated nucleotide precursors. The resulting biotinylated cRNA was fragmented to a size of approximately 50 bp, and approximately 20-30 µg of the biotinylated cRNA was hybridized to the MG_U74Av2 GeneChip (Affymetrix, Santa Clara, CA). The bound cRNA was visualized by binding of streptavidin/phycoerythrin conjugates to the hybridized GeneChip, followed by laser scanning of bound phycoerythrin. Sample_RNA_Isolation: Tissues (~50 mg) were taken frozen and directly solubilized in chaotropic solubilization buffer using a Brinkman Polytron tissue disruptor. Larger tissue fragments (>100mg) were pulverized into frozen powder with a mortar and pestle, pre-chilled to liquid nitrogen temperature, and then the frozen powder was solubilized with the Polytron. RNA was purified using Trizol LS (Life Technologies) and an additional RNA purification step was conducted using the RNAeasy purification kit (Qiagen Inc., Valencia, CA). Approximately 10 µg of purified, total RNA was used for analyses. Sample_sex: male Sample_strain: C57BL6 Keywords = Murine Keywords = Bioinformatics Keywords = mechanical ventilation Keywords = early stress response gene Keywords = LPS
