Quantitative Gene Expression Analysis

Total RNA was isolated from cells comprising the A- and R-SLATEs by standard Trizol (Thermo-Scientific) extraction, according to the manufacturer's protocol. RNA quality was assessed using a NanoDrop 2000 spectrophotometer (Thermo-Scientific) to ensure the 260/280 ratio was within the range 1.8–2.0. Synthesis of cDNA from isolated total RNA was performed using the Maxima First cDNA Synthesis kit (Thermo-Scientific) according to the manufacturer's instructions, in a TcPlus thermocycler (Techne, UK). Quantitative PCR (qPCR) was performed using the default thermal profile of the Eco Real-Time PCR System (Illumina, CA, USA), with the following 40 × three-step cycle: 10 s denaturation, 95 °C; 30 s annealing, 60 °C; and 15 s elongation, 72 °C. The relative expression of genes coding for collagen I and V, keratocan, lumican, decorin, aldehyde dehydrogenase (ALDH) 1 and 3, carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 6 (CHST6), α-smooth muscle actin (αSMA), and fibronectin) was calculated by the comparative threshold cycle (CT) (Eco Software v3.1, Illumina) and normalized to the expression of the POLR2A housekeeping gene (refer to previous work [22] (link) for primer sequences). All experiments were performed three times, independently (n = 3).