RNA extraction and RT‐PCR

Total RNA was extracted from tissues or cultured cells by TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA), after which cDNA was synthesized from 1 μg total RNA using a Reverse Transcription kit (Takara Biotechnology, Shiga, Japan) in accordance with the manufacturer’s instructions. A quantitative PCR was performed with a LightCycler 480 Realtime PCR System (Roche, Mannheim, Germany) using the following protocol: 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. The results were normalized to the expression of β‐actin. The primers used were: ADAMTS7 (forward primer, 5′‐GTGGAGACCCTGGTAGTAGC‐3′ and reverse primer, 5′‐TCTGCGTGGTGCGTGATCTTTA‐3′); BMP2 (forward primer, 5′‐TGACGAGGGTCCTGAGCGA‐3′ and reverse primer, 5′‐CCTGAGTGCCTGCGATACA‐3′); Runx2 (forward primer, 5′‐CTGGTGTCTCGGCTTCAATCT‐3′ and reverse primer, 5′‐TTCAAGGTGCCAAGAGGTAAGT‐3′); Msx2 (forward primer, 5′‐CCGCCTGTGGGACTCTATG‐3′ and reverse primer, 5′‐GGCTGGTACTGCCTTCGTG‐3′); Osteocalcin (OCN) (forward primer, 5′‐AGGGCAGCGAGGTAGTGAA‐3′ and reverse primer, 5′‐TCCTGAAAGCCGATGTGGT‐3′); Osteopontin (OPN) (forward primer, 5′‐AGCAGCAGGAGGAGGCAGAGCACAG‐3′ and reverse primer, 5′‐CTGTGCTCTGCCTCCTCCTGCTGCT‐3′); β‐actin (forward primer, 5′‐ATCTGGCACCACACCTTC‐3′ and reverse primer, 5′‐AGCCAGGTCCAGACGCA‐3′).