Quantitative RT-PCR analysis.
At weeks 2 and 3 after incubation with OIM, total RNAs were isolated using an miRNeasy Mini kit (Qiagen, Hilden, Germany). First-strand cDNAs were synthesized from 1 µg of each total RNA using Super Script VILO Master Mix (Life Technologies, Carlsbad, CA, USA). One µL of each cDNA was subjected to real-time RT-PCR using TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA) for Runx2 (Mm00501584_m1), Osterix (Mm04209856_m1), ALP (Mm04209856_m1), Collagen Type 1 (Col I) (Mm04209856_m1), OPN (Mm00436767_m1) and OC (Mm03413826_m1), and Pre-Developed TaqMan Assay Reagents (Applied Biosystems) for β-actin (Mm02619580_g1) as an internal control. Two-step PCR cycling was carried out as follows: 2 min at 50 °C for 1 cycle, 10 min at 95 °C for 1 cycle, then 15 s at 95 °C and 1 min at 60 °C for 40 cycles. Two independent measurements were averaged, and relative mRNA expression levels were calculated as a ratio to β-actin expression of each sample.