Real-time quantitative PCR analysis of mouse lungs after influenza A infection

Age-matched BALB/c female mice were implanted subcutaneously with slow release estradiol (E2) pellets (35 mg/pellet/mouse) or similar-sized placebo pellets .One week post-implantation, mice were infected intranasally with a sublethal dose of H5N1 A/Vietnam/1203/2004 vaccine reassortant bearing a monobasic HA cleavage site. Mouse lungs were harvested on day 1 post infection and total RNA was extracted using QIAgen RNeasy microarray tissue mini kit. A total of 100 µg high-quality RNA was reverse transcribed using RT2 Easy First Strand Kit (QIAgen). Resultant cDNA was used as the template along with Profiler™ PCR Array Mouse Signal Transduction PathwayFinder™ kit (QIAgen) to quantitate gene expression activated by E2 in response to H5N1 infection. qPCR lung gene expression profiling of estradiol (E2)- or placebo-pellet implanted mice on day 1 post-H5N1 infection as indicated in the summary. A total of 100 ng of high-quality RNA per mouse lung was subjected to gene expression profiling. The experiment was repeated twice with 2 mice per group per assay.