Cycler 480 2

The total RNA was extracted from the tissue samples using TRI reagent (Thermo Fisher Scientific), and cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche) with random hexamer. The resulting cDNA samples were analyzed using SYBR Green on the Roche Cycler 480 II. The PCR primers used for amplifying COL1α1, COL3α1, and TGF-β1 were as follows: COL1α1—F: 5′-aatggtgctcctggtattgc-3′, R: 5′-ggttcaccactgttgccttt-3′; COL3α1—F: 5′-gggatccaatgagggagaat-3′, R: 5′-ggccttgcgtgtttgatatt-3′; and TGF-β1—F: 5′-tgagtggctgtcttttgacg-3′, R: 5′-tgggactgatcccattgatt-3′. The amplification conditions included an initial denaturation at 95°C for 5 minutes, followed by 45 cycles of denaturation at 95 °C for 10 seconds, annealing at 60 °C for 10 seconds, and extension at 72 °C for 10 seconds. Finally, melting curve analysis was performed from 50 to 105 °C with readings taken every 0.5 °C. Statistical comparison of the expression levels of COL1α1, COL3α1, and TGF-β1 was analyzed using paired t-test.

Total RNA was extracted using TRIzol reagent (#17061, iNtRON, Gyeonggi-do, Korea). The RNA samples were adjusted to the same concentration and reverse-transcribed using a high-capacity cDNA reverse transcription kit (#4368813, Applied Biosystems, Waltham, MA, USA). The cDNA samples were analyzed using a Cycler^® 480 II with LightCycler^® 480 SYBRGreenI master mix (#04887352001, Roche Diagnostics, Basel, Swiss), according to the manufacturer’s instructions. The RT-qPCR analysis was performed with an initial denaturation step of 5 min at 95 °C, followed by 45 cycles at 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s. Quantification of RNA values was determined automatically using the LightCycler^® 480 program (Roche Diagnostics, Basel, Switzerland). The RT-qPCR primer sequences used were as follows in the table below (Table 1).