Mm00437419 m1

Total RNA from TA, GCM and QC muscles was extracted in Trizol (Ambion), purified (PureLink RNA mini kit, Invitrogen) and quantified with the spectrophotometer (NanoDrop 1000 Spectrophotometer V3.7).
The Taq Man (Applied Biosystems) gene expression assay was used following the manufacturer’s instructions on triplicate cDNA samples, using the 1X Universal PCR (Life Technologies) master mix and the 1X mix containing specific receptor probes. The following probes were used: nicotinic cholinergic receptor, gamma subunit (AChRγ) (CHRNG; Mm00437419_m1; Life Technologies); insulin growth factor 1 (Igf1; Mm00439560_m1; Life Technologies); interleukin 10 (Il-10; Mm00439614_m1; Life Technologies); Tumor necrosis factor-alpha (TNF-alpha—Mm00443258_m1; Life Technologies). The relative quantification was calculated from the ratio between the number of cycles (Ct) at which the signal exceeded a threshold set within the logarithmic phase of the given gene and that of the reference actin gene (4310881E; Life Technologies). The mean values of the tripled results for each animal were used as individual data for the 2^−ΔΔCt statistical analysis.

RNA was extracted from GCM using TRIzol (Invitrogen) and purified with PureLink RNA columns (Life Technologies). These RNA samples were treated with DNase I and underwent reverse transcription using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies). Real-time PCR analysis was conducted using the TaqMan Gene expression assay (Applied Biosystems) following the manufacturer's recommended protocols. The reactions were performed on triplicate cDNA specimens, employing 1× Universal PCR Master Mix (Life Technologies) and a 1× mix containing specific receptor probes (Life Technologies).
Relative quantification was determined by calculating the ratio between the cycle number (Ct) at which the signal crossed a threshold set within the logarithmic phase of the target gene and that of the reference β-actin gene (4310881E; Life Technologies). The results were used for a 2-ΔCt statistical analysis. The following probes were employed: nicotinic cholinergic receptor, gamma subunit (AChRγ) (CHRNG; Mm00437419_m1; Life Technologies), interleukin 1β (Il-1β; Mm00434228_m1; Life Technologies), interferon-γ (IFNγ) (Mm01168134_m1; Life Technologies).