Quantification of RPE/Choroid Gene Expression

On day 6 following laser-induced CNV and day 11 following NaIO₃-induced retinal degeneration, individual RPE/choroid fractions were isolated from mice and stored at –80°C until use. RNA was isolated using the miRNEASY Kit (Qiagen, Valencia, CA, USA). Absorbance A260/A280 ratio (Take 3 Micro-Volume Plates; Biotek, Winooski, VT, USA) was used to measure the purity of RNA, and samples with measurements of 1.8 to 2.1 were used to generate first-strand cDNA (Qiagen). Using the Realplex 2 Mastercyler (Eppendorf, Hauppauge, NY, USA), PCR amplifications for genes of interest (Table 1) were performed as previously described.⁴⁷ (link) Quantitative values were obtained using cycle number (Ct value) with values normalized to either β-actin or 36B4.⁴⁸ (link)