Total RNA was isolated using the Trizol (Invitrogen, Carlsbad, CA). Single-stranded cDNA was transcribed from the RNA using Promega’s reverse transcription system (Madison, WI). Then PCR was performed with cDNA in a reaction mixture containing 25 mM magnesium chloride (MgCl2), dNTPs, reverse and forward primers, and Taq polymerase (Takara Bio, Shiga, Japan). The following primers were used for PCR: F-COX2, 5′-ATGACTTCCAAGCTGGCCGT-3′ and R-COX2, 5′-CCTCTTCAAAAACTTCTCCACACC-3′ (annealing temperature; 57 °C, PCR product size; 305 base pair): F-RANKL, 5′-GCCAGTGGGAGATGTTAG-3′ and R-RANKL, 5′-TTAGCTGCAAGTTTTCCC-3′ (annealing temperature; 48 °C, PCR product size; 486 base pair): F-OPG, 5′-GGGGACCACAATGAACAAGTTG-3′ and R-OPG, 5′-AGCTTGCACCACTCCAAATCC-3′ (annealing temperature; 55 °C, PCR product size; 409 base pair): F-GAPDH, 5′-CCCCCTACTGCCCACTGCCACCAC-3′ and R-GAPDH, 5′-TCCATCCACTATGTCAGCAGGTCC-3′ (annealing temperature; 52 °C, PCR product size; 420 base pair). The amplified PCR product was electrophoresed on a 2% agarose gel in 1× Tris-Borate-EDTA buffer containing ethidium bromide and visualized using Quantity One software and the Gel Doc 2000 system (Bio-Rad).