Agilent 2100 bioanalyzer series 2

Total RNA from the corneal epithelial cells was extracted in QIAzol reagent (Qiagen NV, Venlo, the Netherlands) and spin-column purified using a RNeasy mini spin column (Qiagen NV). The RNA was extracted from control and irradiated groups, which consisted of six whole individual three-dimensional reconstructed human corneal epithelial models. The quantity and quality of the RNA samples were then measured using UV absorbance (NanoDrop Technologies, Wilmington, DE, USA), and the quality was also assessed using an Agilent 2100 Bioanalyzer Series II (Agilent Technologies, Santa Clara, CA, USA).

Cy3-labeled cRNA samples were synthesized using the Low Input Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer’s instructions. For each time point, 50 ng of total RNA was used to generate first-strand cDNA. After the denaturation (10 minutes at 65°C) and cRNA synthesis (2 hours at 40°C) steps, the reactions were incubated at 70°C for 15 minutes to inactivate the Affinity-Script enzyme (Agilent Technologies). For labeling reactions, cRNA samples were each mixed with 6 μL of transcription master mix cocktail containing Cy3-CTP and incubated at 40°C for 2 hours. Purification was performed using the RNeasy mini spin columns. Labeled cRNA was quantified on a Nanodrop ND-1000 (NanoDrop Technologies), and quality was also checked by Agilent 2100 Bioanalyzer Series II.