Messenger RNA levels for the inflammatory cytokines, TNF-α, INF-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17 and MMP-9, and for the prooxidant redox enzymes, NOX1, NOX2 and NOX4, were determined in corneal and conjunctival epithelium from the 15-month-old wild-type and the M3R−/− mice by real-time PCR. After the euthanasia of mice by CO2 exposure, one eye per mouse was immediately excised and transferred into cooled PBS (Invitrogen, Karlsruhe, Germany) to excise the cornea and conjunctiva under a dissecting microscope. The corneal epithelium was scraped off from the stroma by using fine-point tweezers to obtain corneal epithelial cells. After excision of the conjunctiva, the subepithelial conjunctival tissue was thoroughly removed by Vannas scissors. The isolated epithelia were transferred into 1.5 mL plastic tubes, rapidly frozen in liquid nitrogen and stored at −80 °C. Later, tissue samples were homogenized (FastPrep; MP Biomedicals, Illkirch, France), and the expression of genes was measured by SYBR Green-based quantitative real-time PCR, as previously described [51]. RNA was isolated using peqGOLD TriFast™ (PEQLAB) and cDNA was generated with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Darmstadt, Germany). Real-time PCR reactions were performed on a StepOnePlus™ Real-Time PCR System (Applied Biosystems) using SYBR® Green JumpStart™ Taq ReadyMix™ (Sigma-Aldrich, Steinheim, Germany) and 20 ng cDNA. The relative mRNA levels of target genes were quantified using comparative threshold (CT) normalized to the TATA-binding protein (TBP) housekeeping gene. Messenger RNA expression is presented as the fold-change in the M3R−/− mice versus the wild-type mice. The PCR primer sequences are listed in Table 1.

Primer sequences used for quantitative PCR analysis.

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