Real-time polymerase chain reaction (RT-PCR)

The tibias of all mice were crushed under liquid nitrogen conditions, and RNA extraction was performed following the TRIzol manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). RNA integrity was verified by formaldehyde-agarose gel electrophoresis. 3 µg of total RNA, Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA, USA), and oligo deoxythymidine (dT) (15) primers (Fermentas) were used to synthesize cDNAs (complementary deoxyribonucleic acid) by reverse transcription reactions. Regular PCR was then performed with the first strand of cDNA using a DNA Engine (ABI 7300). We used glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to normalize the data. After denaturing cDNA at 95 °C for 3 min, the cycling conditions were set according to the kit instructions. The PCR primers used in this study are described in Table 1.

OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor kappa-B ligand; COL-I, collagen type I; RUNX-2, Runt-related transcription factor 2; OCN, osteocalcin; SOST, sclerostin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.