Quantitative Real-Time RT-PCR (qPCR) Analysis

Specimens were obtained from ten patients (5 males and 5 females, aged 25–48 years) with keloid and the adjacent normal skin tissues, removed from the subcutaneous tissues, and then cut as much as possible. Total RNA was isolated from the keloid tissues, the adjacent normal skin tissues and the collected normal fibroblasts (NFs) or keloid fibroblasts (KFs) by using TRIzol reagent following the manufacturer’s directions. The RNA concentration (OD260/OD280, ratio1.8–2.0) was then measured by Nanodrop 2000 Spectrophotometer. The High Capacity cDNA Reverse Transcription Kits was used to synthesis the cDNA. The expression levels of target genes were assessed using the primer sequences are shown in Table 1 and performed using the ABI 7500 System (Applied Biosystems). The PCR reaction conditions were as follows: 10 min at 95°C, 40 cycles at 94°C for 15 sec and then 60°C for 1 min. The relative mRNA levels of target genes were determined using the comparative threshold cycle (CT) method using the 2^–ΔΔCT equation. GAPDH was used as an internal control gene. The experiments were repeated five times for each experimental condition.

Primers Used in qPCR