Total RNA was extracted from alveolar macrophages using Qiagen RNeasy Mini Kit (Valencia, CA) and treated with Qiagen DNase I to eliminate contaminating genomic DNA. cDNA was synthesized using reverse transcription with 1 μg of total RNA in total volume of 10 μl per reaction in Bio-Rad iScript cDNA synthesis kit (Hercules, CA). Quantitative PCR was carried on the Bio-Rad iCycler. Amplification was performed in Bio-Rad iQ SYBR green supermix containing specific primers and with denaturing at 95°C for 20 seconds, annealing at 58°C for 20 seconds, and extension at 20 seconds. Samples were run in triplicate. QuantumRNA class II 18S primers were purchased from Ambion (Austin, TX). PCR amplicons from all species were normalized for the amount of 18S in the same cDNA sample. Dilution curves showed that the real-time PCR efficiency was greater than 95% for all genes analyzed. Real-time SYBR green dissociation curves showed one species of amplicon for each primer set. All primers were designed in our laboratory and were obtained from Invitrogen.
