Il 1β mm00434228

Total RNA was isolated from snap frozen liver samples (RNeasy Plus Mini Kit, Qiagen, Germany). The quantity of RNA was measured on Colibri Microvolume Spectrometer (Titertek-Berthold, Germany). 500 ng of total RNA was used to be reversely transcribed into cDNA using High-Capacity cDNA Reverse Transcriptase Kit (ThermoFisher, Germany) according to the manufacturer’s instructions. Each qRT-PCR reaction was performed using 2 µl of cDNA in a final volume of 10 µl. All samples were run in duplicates. RT-PCR was performed using the following TaqMan Gene Expression Assays: il-1β Mm00434228, tnf-α Mm00443258, ifn-γ Mm01168134, il-12a Mm00434169, il-12b Mm00434174_m1, il-4 Mm00445259, acta-2 Mm00725412, il-13 Mm00434204, il-10 Mm01288386 (ThermoFisher, Germany). Cycling was performed using QuantStudio 3 (Thermo Fisher Scientific, Germany) under the following reaction conditions: 50°C for 2 min followed by 95°C for 10 min, 45 cycles at 95°C for 15 s, and at 60°C for 1 min. The ΔΔCt method was utilized for relative quantification (16 (link)). Gene expression values were normalized to the endogenous reference gene GAPDH (Rodent GAPDH control reagent, ThermoFisher, Germany) and presented as normalized expression values relative to naive controls.

At 52 weeks post-infection, total RNA was isolated from snap frozen liver samples (RNeasy Plus Mini Kit, Qiagen, Hilden, Germany) and reversely transcribed into cDNA using High-Capacity cDNA Reverse Transcriptase Kit (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. RT-PCR was performed using the following TaqMan Gene Expression Assays: IL-1β Mm00434228, IL-4 Mm00445259, IL-6 Mm00446190, IL-10 Mm01288386, IL-12α Mm00434169, IL-13 Mm00434204, IFN-γ Mm01168134, Acta-2 Mm00725412, Col1-α-2 Mm00483888 (ThermoFisher Scientific, Waltham, MA, USA). Cycling was performed using QuantStudio 3 under the following reaction conditions: 50°C for 2 min followed by 95°C for 10 min, 45 cycles at 95°C for 15 s, and at 60°C for 1 min. Gene expression values were normalized to the endogenous reference gene gapdh (Rodent GAPDH control reagent, ThermoFisher Scientific, Waltham, MA, USA) and presented as normalized expression values relative to naive controls.