Autoimmunity rt2 profiler pcr array

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)].

In this case, qPCR was performed to analyze the commitment ability of the cells, the miRNA content inside the cells and the miRNA content of the exosomes (extracellular miRNA). Exosomal miRNA expression profiles were analyzed using miScript miRNA PCR Arrays (MIHS-001Z, Qiagen, Hilden, Germany) that investigated the expression of the 84 most abundantly expressed human miRNAs. The cDNA was synthesized from the total RNA using the miScript II Reverse Transcription Kit (Qiagen, Hilden, Germany), according to the user manual. The cDNA was pre-amplified with the miScript PreAMP PCR Kit, then mixed with QuantiTect SYBR Green PCR Master Mix, miScript Universal Primer, and RNase-free water. Real-time PCR was performed according to the user’s manual on inflammatory response and Autoimmunity RT2 profiler PCR Array (Qiagen, Hilden, Germany) 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, Hilden, Germany).