2.5. Reverse transcription‐polymerase chain reaction (RT‐PCR)

Total RNA was extracted using mRNA isolation kit according to the manufacturer's instructions (Takara). In RT‐PCR, an RNA population was converted into cDNA by reverse transcription (RT), and then, the cDNA was amplified by PCR. The sequences of the primers used were as follows: runt‐related transcription factor 2 (Runx2) (mice), CCAGGCAGGTGCTTCAGAACTG (forward), GGTAGTGAGTGGTGGCGGACAT (reverse); osteocalcin (OCN) (mice), GCTACCTTGGAGCCTCAGTC (forward), ATGCGTTTGTAGGCGGTCTT (reverse); Runx2 (human), CCCAGGCAGTTCCCAAGCATTT(forward), GGTAGTGAGTGGTGGCGGACAT (reverse); OCN (human), GGCAGCGAGGTAGTGAAGAGAC (forward), GGTCAGCCAACTCGTCACAGTC (reverse); bone morphogenetic protein‐2 (BMP‐2) (human), GGTCCTGAGCGAGTTCGAGTTG (forward), TGACCTGAGTGCCTGCGATACA (reverse). Quantitative RT‐PCR (RT‐qPCR) was performed with SYBR green fluorescence (Bio‐Rad Laboratories Inc). β‐actin, a housekeeping gene, was used for internal normalization. Gene expressions were analysed using the 2–ΔΔCt method.

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