Cytokine and Growth Factor Expression Analysis in Pseudomonas Infection

After infection with PA for 24 h, total RNA was isolated using a GeneJET RNA Purification Kit (Thermo Scientific, Waltham, MA, USA, K0732). RNA concentrations in each sample were assessed using a Nanodrop (Thermo Scientific) and cDNA was synthesized using a Maxima^TM H Minus cDNA Synthesis Master Mix with a dsDNase kit (Thermo Fischer, Waltham, MA, USA, K1652). RT-qPCR was performed on an Applied Biosystems StepOnePlus using the TaqMan primers Interleukin-8 (Hs00174103_m1, CXCL8), Interleukin-6 (Hs00174131_m1, IL-6), Interleukin 1-β (Hs01555410_m1, IL-1β), FGFR1 (Hs00241111_m1, FGFR1), FGFR2 (Hs01552918_m1, FGFR2), FGFR3 (Hs00179829_m1, FGFR3), FGFR4 (Hs01106910_g1, FGFR4), Transforming growth factor-beta (Hs00998133_m1, TGFB1), klotho (Hs00934627_m1, KL), and reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Fold changes were calculated via the ΔΔCT method previously described [23 (link)].