Quantifying Osteogenic Gene Expression

Total RNA was extracted with TRIzol (Invitrogen, Carlsbad, USA) and then converted to cDNA with Hifair II 1st Strand cDNA Synthesis Kit (Yeasen, Shanghai, China) following the kit instructions. SYBR green PCR Master Mix (Takara, Dalian, China) was used for real-time qPCR on an MX 3000 platform (Agilent, Boeblingen, Germany). The primers for real-time qPCR were as follows: Runx2, 5’-ATAGCAAAGGCCCTCACTAA-3’ (forward) and 5’-AACTGGCTCTTCTGCTGATT-3’ (reverse); Col 1, 5’-GAGGCATAAAGGGTCATCGTGG-3’ (forward) and 5’-CATTAGGCGCAGGAAGGTCAGC-3’ (reverse); OC, 5’-TGACCTCACAGATGCCAAGC-3’ (forward) and 5’- CGCCGGAGTCTGTTCACTAC-3’ (reverse); and GAPDH, 5’- ACCACAGTCCATGCCATCAC-3’ (forward) and 5’-TCCACCACCCTGTTGCTGTA-3’ (reverse).
The expression levels of each target gene were normalized to the corresponding GAPDH threshold cycle (CT) values using the 2−▵▵CT comparative method [16 (link)].