In this study we utilized a genome-wide approach to analyze circadian patterns of gene expression in mouse lung lungs, both in the basal state and in the setting of systemic inflammation caused by endotoxemia. We chose the lung because it represents a primary portal for systemic infection and organ failure in critically ill patients, and because the lung exhibits strong physiological circadian rhythms in health and in diseases such as asthma. The gene expression the data presented here was correlated to histological observations and metabolite measurements derived from the same biological samples, in order to obtain a broad picture of how inflammation impacts circadian rhythms in mouse lung.
Overall design
To examine circadian regulation in mouse lung we performed 2 independent time-series experiments (Microarray Experiments #1 and #2), in which groups of mice were euthanized at 4 hour intervals for 48 hours. The purpose of Microarray Experiment #2 was to compare circadian gene expression in healthy lungs (samples labeled with the prefix “NT”) to lungs derived from endotoxemic animals (samples labeled with the prefix “LT”). For Microarray Experiment #2, 94 mice were placed under constant light conditions (LL 12:12, food ad libitum) at CT12 the prior day (7:00 PM local time). At CT10 (5:00 PM) on the day of the experiment a sub-group of 40 mice received a single intraperitoneal injection of 12 mg/kg E. coli O127:B8 endotoxin (LPS, Sigma L3129, Lot 029K4055), and sample collection began directly after. 3-4 mice per group were sacrificed at consecutive 4 hour intervals for 2-3 days. The left lung and the right upper lobe were frozen immediately in liquid nitrogen for microarray analysis. For mRNA isolation lung tissue was immersed in RNAlater (Qiagen) and total RNA was then extracted using the RNeasy Mini Kit (Qiagen). RNA quality (RIN>=7) was confirmed using an Agilent 2100 Bioanalyzer. RNA from all biological samples was labeled at once using the Ambion TotalPrep-96 RNA Amplification Kit. The samples were then blinded, randomized to chip position and hybridized to the Illumina MouseRef-8 v2.0 Expression BeadChips. For Microarray Experiment #2 at the Channing Division of Network Medicine (Brigham and Women’s Hospital). Two biological samples (NT00L1 and LT00L1) were represented by 2 technical replicates on the microarray. The remaining biological samples were represented by a single technical replicate.
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