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 gene expressions of interferon (IFN) α, IFNγ, TNFα, interleukin (IL) 6, IL-1α, CXCL8, CCL5, CXCL10, CCL3, CCL2 and TLR3 were detected by RT-qPCR. 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 housekeeping gene β-actin, and the results are expressed as arbitrary units. The primer sequences and their publication sources are presented in Table 1 . Primer sequences for the β-actin, IL-1α and CCL3 genes were designed as described previously [31] . The ferret-specific genes are available in the NCBI nucleotide database [32] . The designed primer sequences and the GenBank accession numbers are presented in Table 1 . The amplification products were detected by electrophoresis to validate the sizes of the product in accordance with the primer design, and the products were purified using a QIAquick PCR Purification Kit (Qiagen). Sequencing reactions were performed with ABI Prism BigDye Terminator Cycle Sequencing v.3.1 Ready Reaction (Applied Biosystems) and analysed using an ABI PRISM model 3730 automated sequencer (Applied Biosystems). The amplified sequences correlated with the ferret specific target sequences.