18s universal primer pair

Eyes were rapidly enucleated and placed into a sterile Petri dish resting on ice. The RPE/choroid complex was promptly separated from neuroretina. Both tissue complexes were processed for RNA using RiboZol, followed by treatment with DNase I (catalog number AMPD1; Sigma-Aldrich) to remove any contaminating genomic DNA. The DNasetreated RNA was then converted into cDNA using iScript Reverse Transcription Kit (catalog number 170-8841; Bio-Rad, Mississauga, Canada). PCR primers targeting rat were designed using National Center for Biotechnology Information Primer Blast; detailed sequences are summarized in Table 1. Quantitative analysis of gene expression was generated using an ABI 7500 Real-Time PCR (Applied Biosystems, Foster City, CA) sequence detection system and the SYBR Green Master Mix Kit (catalog number 1725271; BioRad). Gene expression was calculated relative to 18S universal primer pair (catalog number AM1718; Ambion, Waltham, MA) expression using the DC T method. Data were quantified by DDC T method.

Eyes were rapidly enucleated and placed into a sterile petri dish resting on ice. The RPE/choroid layer was promptly separated from the neuroretinal layer. Both layers were processed for RNA using TRIzol (Invitrogen, Carlsbad, CA), followed by treatment with DNase I (Qiagen, Hilden, Germany) to remove any contaminating genomic DNA. The DNase-treated RNA was then converted into cDNA using M-MLV Reverse Transcriptase (Invitrogen). PCR primers targeting rat IL-1b, tumor necrosis factor-a, IL-6, IL-17, NLRP-3 (NACHT, LRR and PYD domains-containing protein 3), VEGF, pigment epitheliumederived factor, fibroblast growth factor, and platelet-derived growth factor were designed using National Center for Biotechnology Information Primer Blast (Table 1). Quantitative analysis of gene expression was generated using an ABI Prism 7700 sequence detection system and the SYBR Green Master Mix Kit (BioRad, Hercules, CA). Gene expression was calculated relative to 18S universal primer pair (Ambion, Waltham, MA) expression using the DCt method.