Chicken brain and lung gene expression profiles following infection with two recombinant H5N3 avian influenza viruses - rH5N3 Ori (P0) and rH5N3 P6

Chicken brain and lung gene expression profiles following infection with two recombinant H5N3 avian influenza viruses - rH5N3 Ori (P0) and rH5N3 P6 Groups of 6 specific pathogen free Leghorn chickens were infected oronasally with 4.0 x 10e4 pfu of rH5N3 Ori, 4.0 x 10e4 rH5N3 P6, or sterile PBS. At 1 and 3 days post-inoculation 3 birds from each group were humanely euthanized and RNA extracted from brain and lung samples. 100 mg of each tissue was homogenized in 1 ml of TriPure isolation reagent with 1.0 mm zirconia/silica beads. This was followed by the addition of 0.2 ml chloroform, thorough vortexing and phase separation by centrifugation. The aqueous phase was mixed with an equal volume of 75% ethanol and passed through an RNeasy column (Qiagen). The column was washed once with RW1 and twice with RPE buffer before eluting the RNA in nuclease free water. OD260, OD280 and RNA concentration were determined on a NanoDrop ND-1000 spectrophotometer. RNA concentration and integrity were further assessed using a eukaryotic total RNA nanochip and an Agilent Bioanalyzer 2100.