At designated timepoints after the infectious challenge, the mice were anesthetized and sacrificed. To reduce the gene expression signal of circulating leukocytes in the pulmonary vasculature, the pulmonary vasculature was exsanguinated and the pulmonary arterial system was flushed with PBS. The lungs were also submitted to repeated BAL to reduce the leukocyte burden of the airspaces. The lungs were then excised, homogenized, and total RNA was isolated using the RNeasy system (Qiagen, Valencia, CA). cRNA was synthesized, then amplified, from equal masses of total RNA extracted from the lungs of infected/sham challenged mice using the Ilumina TotalPrep RNA amplification kit (Ambion, Austin, TX).
Label
Cy3
Label protocol
Hybridized cRNA was labeled for 10 minutes with Cy3.
Hybridization protocol
Amplified cRNA was then hybridized and labeled on Sentrix Mouse-6 Expression BeadChips (Illumina, Inc., San Diego, CA).
Scan protocol
All microarrays were scanned on a BeadStation 500 (Illumina).
Description
Sham 3: PBS exposed
Data processing
Data were background corrected using the RMA method, then transformed by taking the base-two logarithm, and quantile normalized. Analysis of the microarray output was performed using a one-way ANOVA to identify infection-induced changes. P values were modeled using a beta-uniform mixture (BUM) model (30), and combined with false discover rate to determine a cutoff of p values. The analysis program was written in R (R Foundation for Statistical Computing, Vienna, Austria), utilizing the Illumina library developed by Simon Lin and Pan Du, Northwestern University.
