2.1.4. Osteogenesis-Related Gene Expression of MC3T3 Cells

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed to determine the osteogenic gene expression of MC3T3. Cells were seeded at 1.5 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/well under different concentrations of glucose and curcumin. The total RNA from all groups was first extracted using the RNAiso plus kit (Takara Bio, Japan), determined with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, NC, USA), and was reversed to cDNA by the Reverse Transcription Kit (Takara Bio, Tokyo, Japan). The RT-qPCR was performed using a SYBR green kit and specific primers (Dalian Bao Biological Takara Corporation, Dalian, China) via the StepOnePlus™ Real-Time PCR system (Thermo Fisher Scientific, Shanghai, China). Primers are as follows: Runx2, forward 5'-CATTTGCACTGGGTCACACGTA-3', reverse 5'- GAATCTGGCCATGTTTGTGCTC-3' (159 bp); *Opn* forward 5'- TACGACCATGAGATTGGCAGTGA-3', reverse 5'- TATAGGATCTGGGTGCAGGCTGTAA-3' (127 bp); Col-1 forward 5'- GTGGCGGTTATGACTTCAGC-3', reverse 5'-TCACGAACAACGTTAGCATC-3' (154 bp); GAPDH forward 5'-TTCGACAGTCAGCCGCATCTT-3', reverse 5'- ATCCGTTGACTCCGACCTTCA-3' (145 bp). The PCR reactions were activated at 95 ◦C for 30 s, followed by an amplification target sequence of 40 cycles at 95 ◦C for 5 s, 60 ◦C for 34 s, and 95 ◦C for 15 s. The relative expression levels of the genes were calculated by the 2−ΔΔCT method.
