Sybr green 1

TRIzol^® reagent (Thermo Fisher Scientific, Inc.) was used to extract intracellular total RNA, and a Prime Script RT kit (Takara Bio, Inc., Otsu, Japan) was used to synthesize single-stranded cDNA. qPCR was performed using an ABI 7500 real-time PCR system (Thermo Fisher Scientific, Inc.). qPCR was conducted using cDNA (1 µl) with SYBR Green-1 (20 µl; Takara Bio, Inc.) according to the manufacturer's protocol under the following conditions: Activation at 95°C for 10 min, then 40 cycles of amplification (95°C for 10 sec, 60°C for 24 sec and 72°C for 20 sec) and a final extension at 72°C for 1 min. All the reactions were performed in triplicate and target gene expression was normalized to the reference gene β-actin. The 2^−Δ∆Cq method (26 (link)) was applied to calculate the relative expression level of the target genes. The PCR products were subjected to melting curve analysis and a standard curve to confirm the correct amplification. The primers used for PCR were as follows: Cathepsin K (CTSK), forward 5′-AGAACGGAGGCATTGACTCT-3′, reverse 5′-GATGGACACAGAGATGGGTC-3′; TRAP, forward 5′-CGATCACAATCTGCAGTACC-3′, reverse 5′-ACCCAGTGAGTCTTCAGTCC-3′; nuclear factor of activated T cells cytoplasmic 1 (NFATc1), forward 5′-CGCAAGTACAGTCTCAATGG-3′, reverse 5′-CAGGTATCTTCGGTCACACT-3′; and β-actin, forward 5′-AGGCCAACCGTGAAAAGATG-3′ and reverse 5′-TGGCGTGAGCGAGACCATAG-3′.