Syber premix extaq

Total RNA was extracted from PBMCs, PFMCs, and ESCs using Trizol solution (Qiagen) based on the manufacturer's protocol. Quantity and purity of the extracted RNA were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific), and RNA integrity was assessed by electrophoresis on 2% agarose gel. For cDNA synthesis, 1 μg of RNA was reverse transcribed into cDNA using a Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) according to the protocol. The qRT‐PCR was performed in duplicate using Rotor‐Gene 3000 (Corbett Research) with the SYBER premix Extaq (Biofact). The mRNA expression of MCP‐1, HGF, and IGF‐1 were normalized using glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) mRNA as a housekeeping gene. The sequences of the primers and size of amplicons are shown in Table 1. Briefly, 10 μl SYBER premix Extaq (Biofact), 1 μl primer pairs, 1 μl cDNA template, and 8 μl DNase‐free water were amplified in Rotor‐Gene 3000 with cycling conditions as 95°C step for 15 min (initial denaturation and activation of enzyme), followed by 40 cycles of 95°C for 20 s, annealing and elongation at 60°C for 40 s and the melting step at 60 to 99°C. All reactions were run in duplicate.