Heterogeneous pathological outcomes after experimental pH1N1 influenza infection in ferrets correlate with viral replication and host immune responses in the lung

Vidaña, Beatriz; Martínez, Jorge; Martínez-Orellana, Pamela; García Migura, Lourdes; Montoya, María; Martorell, Jaime; Majó, Natàlia

The expression of the proapoptotic genes caspase 8 (CASP8) and BAX were quantified by RT-qPCR. The primer sequences are presented in Table 1 . The RT-qPCR techniques were performed as previously described [31] . Briefly, RNA extraction was performed on the ferret lung tissue samples with an RNeasy Mini Kit using the RNA stabilisation and on-column DNase digestion protocols (Qiagen). Reverse transcription was performed using an ImProm-II reverse transcription system (Promega) at 0.5 μg RNA. PCR was performed using a Power SYBR green kit (Applied Biosystems) and Fast 7500 equipment (Applied Biosystems). PCR reactions were performed in 10 μL reaction volumes using the Power SYBR green kit (Applied Biosystems); 40 amplification cycles were used, and the annealing temperature was 60°. The expression levels were normalised using the house-keeping gene β-actin, and the results are expressed as arbitrary units. Primer sequences of CASP8 and BAX were designed as described previously [31] . The designed primer sequences and the GenBank accession numbers are presented in Table 1 . Primer validation was performed as described above in the cytokine gene expression profile section.