Gene Expression Analysis in Bone Healing

Since the tooth sockets of mouse upper molar tooth are very small, we used the left maxilla including extraction sockets for RNA isolation. In the semistabilized femoral fracture model, the whole femur including fracture site was subjected to RNA isolation as described previously (Mohan et al., 2005 (link)). Total RNA was isolated from these bones as previously described (Yang et al., 2017 (link)). The maxilla and femur samples were dissected on days 0, 3, 5, and 7 after the operation. Quantitative real-time PCR was performed by using the One-Step SYBR Prime Script PLUS RT-PCR (TAKARA, Shiga, Japan) using Step One Plus or 7500 Fast systems (Thermo Fisher Scientific). The mRNAs examined were Sp7 (Osterix), Col1a1 (type I collagen), Runx2, Sox9, Acan (Aggrecan), Col2a1 (type II collagen), and Col10a1 (type X collagen). Relative expression was determined using Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Expression levels were calculated as fold-change relative to control group (0 days), which were isolated immediately after the operations. The primers used for each gene are shown in Table 1 in Supplemental information.