2.3. Real-Time Reverse Transcription Polymerase Chain Reaction

After the experimental treatment of the HBEpCs and A549 cells total RNA was extracted from these cells with the RNeasy plus Mini Kit (Qiagen, Germantown, MD, USA). RNA was quantified with a NanoDrop spectrophotometer (Thermofisher Scientific, Foster City, CA, USA). Total RNA was then reverse transcribed with the High-Capacity complementary DNA (cDNA) Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). All reverse transcription-polymerase chain reactions (RT-PCRs) were carried out using standard TaqMan primers for NFKBIB (assay id # Hs00182115_m1), NF-κB (assay ID# Hs00765730_m1), and glyceraldehyde phosphate dehydrogenase (GAPDH) (assay ID # Hs03929097_g1), which were purchased from Applied Biosystems (Thermofisher Scientific). Fold change in expression was determined using the ∆∆ct method after normalizing to GAPDH [22].

miRNA was isolated using the miReasy Kit (Qiagen) and analyzed by RT-PCR with the TaqMan MicroRNA Reverse Transcription Kit (Lifetechnologies, Foster City, CA, USA) and specific primers for miR-4776 (assay # 462695), 4514 (assay # 462737), 4742 (assay # 463053) and the U6 (assay # 001973) control. Influenza matrix gene expression was quantified and reported as influenza copy number. RT-PCR was performed using TaqMan assay with matrix-specific primers, as reported earlier [24]. The M segment of the RT-PCR was specific for viral RNA. A standard curve was generated from the cloned influenza IAV matrix gene by RT-PCR for IAV quantification.