ONH Dissection and RNA Extraction

Following euthanasia, enucleated globes were rapidly chilled in cold phosphate-buffered saline (PBS) and the anterior segment, lens, and retina removed. The posterior segment was laid flat on chilled wax and the ONH trephined out with a 1.5-mm biopsy punch (Acuderm, Ft. Lauderdale, FL, USA). After removing the remaining sclera and nerve sheath, the ONH was positioned in a custom-designed mold to allow separation of the anterior 0.4-mm ONH (Supplementary Fig. S1). Dissected ONHs were frozen in dry ice and stored immediately at –80°C until all samples were collected and ready for RNA extraction.

Prior to RNA extraction, all ONHs were randomized into RNA extraction groups (about eight ONHs per group) to limit possible batch effects. RNA was extracted with the MiRNEasy kit (Qiagen, Venlo, Netherlands) in accordance with the manufacturer's guidelines and as previously described.²⁵ The OHSU Gene Profiling Shared Resource unit analyzed total isolated RNA quality (RNA integrity number or RIN score) and measured concentration with the Agilent Bioanalyzer Pico Chip, Santa Clara, CA, USA. RIN scores and RNA concentrations (mean of 61–64 ng) were similar among naïve, PT-CEI, and normotensive CEI ONH.