Relative tumour necrosis factor-α (TNF-α), nuclear factor kappa beta (NFK-ß), and interleukin-6 (IL-6) messenger RNA (mRNA) expression analyses were performed using StepOne Plus Real-Time polymerase chain reaction (PCR) system technology (Applied Biosystems, Foster City, CA, USA) using synthesised cDNA from rat kidney RNA. A quantitative PCR was run using a TaqMan probe mix based on TaqMan probe-based technology (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed using primers generated for rat TNF-α Rn00562055_m1, rat NFK-ß Rn01399583_m1, rat IL-6 Rn01410330_m1, and rat β-actin Rn00667869_m1. The results are expressed as relative-fold, compared with control animals. The expression data for β-actin in each tissue were used as the endogenous control. Each determination was performed in triplicate for each tissue in a 96-well optical plate for both targets, using 9-μL cDNA (100 ng), 1 μL of Primer Perfect Probe mix, and 10 μL of QuantiTect Probe PCR Master Mix (Qiagen) in each 20-μL reaction. The plates were heated for 2 min at 50 °C and then 10 min at 95 °C. Subsequently, 40 cycles of 15 s at 94 °C and cycles of 60 s at 60 °C were conducted. All data are expressed as the fold-change in expression compared with the expression in other animal groups, using the 2-delta-delta Ct (2-ΔΔCt) method [13 (link), 14 (link)].
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