Quantifying Gene Expression in Scar Tissue

The scar tissue was stored at −80 °C. In total, 20 mg of scar tissue was ground in liquid nitrogen. Total RNA was extracted from the scar tissue using the SV Total RNA Isolation System (Bio-Rad CFX96 system, Bio-Rad, Hercules, CA, USA). The purity of RNA was evaluated by the ratio of 260 nm/280 nm absorption. The PrimeScript^TM RT Reagent Kit (TaKaRa, Beijing, China) was used to synthesize cDNA by reverse transcription, and 2 μg of total RNA was reverse transcribed by the Oligo (dT) 15 Primer (TaKaRa, Beijing, China). The cDNAs from the internal reference gene and genes of interest were PCR amplified in an Agilent Mx3000P Real-Time PCR System using TB Green^TM Premix Ex Taq^TM II (TaKaRa, Beijing, China). Primer sequences were designed using Primer Premier 6 (Applied Biosystems) and all primers were synthesized by Sangon Biotech (Shanghai, China). The information about the primers is shown in Table 1. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal reference. The relative expression levels were calculated using the 2^−∆∆CT method [23 (link)].