Quantitative RT-PCR Analysis of Conjunctival Gene Expression

Total RNA from conjunctiva or the corneal epithelium was extracted using a QIAGEN RNeasy Plus Micro RNA isolation kit (Qiagen) following the manufacturer’s protocol. Conjunctiva was surgically excised. One sample equaled the tissue pooled from both eyes of each animal. After isolation, the concentration of RNA was measured and cDNA was synthesized using the Ready-To-Go™ You-Prime First-Strand kit (GE Healthcare) as previously described15 (link). Real time PCR was performed using specific Taqman probes for IL-17A (Il17a, Mm0043918_m1), IFN-γ (Ifn-γ, Mm00801778_m1), IL-13 (Il13, Mm99999190_m1), Foxa2 (Foxa2, Mm00839704_mH), Integrin α2 (Itga2 or DX5, Mm00434371_m1) (Taqman Universal PCR Master Mix AmpErase UNG) in a commercial thermocycling system (StepOnePlus™ Real-Time PCR System, Applied Biosystems), according to the manufacturer’s recommendations. The beta-2 microglobulin (β2 m) (B2m) (Mm00437762_m1) gene was used as an endogenous reference for each reaction. The results of quantitative PCR were analyzed by the comparative C_t method in which the target of change = 2⁻ΔΔ^Ct and were normalized by the C_t value of β2 m and the mean C_t of relative mRNA level in the untreated naïve group. Gene expression of 3–4 mice per group per time point in two independent experiments with a total of 6–8 mice was determined.