Fam dye labeled primers

Total RNA was extracted from corneal or conjunctival tissue using a commercial reagent (TRIzol; Invitrogen, Carlsbad, CA) and an RNA purification kit (RNeasy Micro Kit; Qiagen, Germantown, MD).First-strand cDNA was synthesized with random hexamers using reverse transcriptase (SuperScript III; Invitrogen), and quantitative real-time polymerase chain reaction (PCR) was performed using predesigned primers (Taqman PCR Mastermix and FAM dye-labeled primers; Applied Biosystems, Foster City, CA) for TSP-1 (Mm00449032_g1), IL-1β (Mm00434228_m1), IL-6 (Mm00446190_m1), IL-23 ((Mm00518984_m1), IL-17A (Mm00439619_m1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Mm99999915_g1). The GAPDH gene was used as the endogenous reference for each reaction. The results were analyzed by the comparative threshold cycle (CT) method using a commercial analysis software (LightCycler, version 3; Roche Diagnostics Corp., Indianapolis, IN).

The corneal epithelial tissue was treated with Guanidinium thiocyanate (TRIzol; Invitrogen Inc, Carlsbad, CA) for extraction of total RNA, and purified using RNeasy Micro Kit (Qiagen Inc., Germantown, MD). The first-strand of cDNA was synthesized by random hexamers using reverse transcriptase (SuperScript III; Invitrogen Inc., Carlsbad, CA), and subsequently predesigned primers (Taqman PCR Mastermix and FAM dye-labeled primers; Applied Biosystems Inc, Foster City, CA) were used to perform RT-PCR for Serpinf1 (Mm00441267_g1), IL-6 (Mm00446190_m1), IL-1β (Mm00434228_m1), IL-17A (Mm00439619_m1), IL-23 (Mm00518984_m1). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Mm99999915_g1) gene was used as the endogenous reference for each reaction. The results of RT-PCR were analyzed using the comparative threshold (CT) cycle method using the commercial analysis software (LightCycler, version 3; Roche Diagnostics Corp., Indianapolis, IN).