Real-time PCR Analysis of Osteoclast Markers

Real-time PCR was performed as reported previously [41 (link)]. Briefly, total RNA was extracted using TRI-solution (Bioscience; Seoul, Republic of Korea) following the manufacturer’s instructions. Single-stranded cDNA was synthesized from 1 μg of total RNA using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). We performed real-time PCR using a LightCycler 1.5 real-time PCR system (Roche Diagnostics, Basel, Switzerland) and the SYBR Premix Ex Taq (Takara Bio Inc., Shiga, Japan). The primer sequences used were as follows: TRAP (Acp5), 5′-TCCCCAATGCCCCATTC-3′ and 5′-CGGTTCTGGCGATCTCTTTG-3′; Cathepsin K (Ctsk), 5′-GGCTGTGGAGGCGGCTAT-3′ and 5′-AGAGTCAATGCCTCCGTTCTG-3′; Dcstamp, 5′-CTTCCGTGGGCCAGAAGTT-3′ and 5′-AGGCCAGTGCTGACTAGGATGA-3′; Nfatc1, 5′-ACCACCTTTCCGCAACCA-3′ and 5′-TTCCGTTTCCCGTTGCA-3′.