Mm01168134 m1

Inventoried TaqMan assays were purchased from Life Technologies [Mm00443258_m1 (Tnf), Mm00812512_m1 (Prf1), Mm01168134_m1 (Ifng), Mm00442837_m1 (Gzmb), Mm01182107_g1 (Cd8a), Mm00445235_m1 (Cxcl10), Mm00434946_m1 (Cxcl9), Mm00492586_m1 (Ido1), Mm00439531_m1 (Stat1)]. All qRT-PCR reactions were performed using the 7900HT Fast Real-Time PCR system and Taqman gene expression master mix (Applied Biosystems) with a standard cycling program of 40 cycles at 95°C for 15 s and at 60°C for 1 min. All reactions were run in triplicate and normalized to human 18S (Hs99999901_s1). Data were analyzed using the 2^-ΔΔCT method.

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).