PrimeScript™ RT Master Mix (TAKARA) was used to extract the total RNA (0.5 μl) of CTRL, NOR, TF and SF group samples and convert it to cDNA with a total capacity of 10 μl. Quantitative PCR was performed using the BioRad CFX96 Touch Real-Time Quantitative PCR system. The final reaction volume was 25 μl, and each sample used a 2 μl cDNA template. GAPDH acted as an internal control. The reaction was repeated 3 times, with at least 3 independent runs. Data analysis was performed using the ΔΔCt method Bio-Rad Ranking Manager 3.1. Primer sequences are shown in Table 1 .Table 1
Primer sequences used for the validation amongst upregulated genes.
| Name | Direction | 5’→3′ |
|---|---|---|
| COL1A1 | Forward | GCTCCTCTTAGGGGCCACT |
| Reverse | ATTGGGGACCCTTAGGCCAT | |
| RUNX2 | Forward | GACTGTGGTTACCGTCATGGC |
| Reverse | ACTTGGTTTTTCATAACAGCGGA | |
| GAPDH | Forward | AATGGATTTGGACGCATTGGT |
| Reverse | TTTGCACTGGTACGTGTTGAT |