RNA Extraction and cDNA Preparation

Corneal tissue was homogenized in Precellys24 high-throughput tissue homogenizer (Bertin Corp., Rockville, MD, USA) using three 30-second cycles of homogenization at 5500 rpm with 2.8-mm ceramic beads. Total RNA was extracted using NucleoSpin RNA/Protein isolation kit (Macherey-Nagel, Inc., Bethlehem, PA, USA). Between 1 and 4 ug high-quality RNA was extracted from approximately half a button as a starting material as measured by NanoDrop 2000c (Thermo Scientific, Wilmington, DE, USA). Extracted RNA underwent quality assessment on Agilent 2100 Bioanalyzer (Thermo Scientific) by visual examination of ribosomal bands and RIN (RNA Integrity Number) calculation. RNA was reverse transcribed to cDNA using RT² First Strand Kit (SABiosciences, Qiagen, Valencia, CA, USA).