Quantifying Gene Expression in Tissue Engineered Constructs

Total RNA was isolated from cells grown onto scaffolds or onto plastic for 7 days using the total RNA purification Plus kit (Norgen Biotek, Toronto, ON, Canada). The RNA quality and concentration of the samples were measured with the NanoDrop™ ND-1000 (Thermo Fisher Scientific). For each sample, 500 ng of total RNA was reverse-transcribed using an RT2 First Strand kit (Qiagen, Hilden, Germany) in a final reaction volume of 20 μL [39 (link)]. Real-time PCR was performed according to the user’s manual on wound healing or inflammatory response and Autoimmunity RT2 profiler PCR Array (Qiagen) or the primer reported on Table 1 with a StepOnePlus™ Real-Time PCR System (Applied Biosystems™, Foster City, CA, USA) using RT2 SYBR Green ROX FAST Master Mix (Qiagen). Thermal cycling and fluorescence detection were as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. At the end of each run, a melting curve analysis was performed using the following program: 95 °C for 1 min, 65 °C for 2 min with optics off, and 65 °C to 95 °C at 2 °C/min with optics on. Each experiment was repeated 3 times, and each measure was repeated 3 times [55 (link)].