Quantitative Analysis of Gene Expression in Bone Tissues

Bone tissue samples were harvested from mice, snap-frozen in liquid nitrogen, and ground into powder using a hammer. The powdered bone tissue samples were transferred into RNAse-free microcentrifuge tubes and RNA was extracted using TRIzol reagent (Cat#. 15596026, Invitrogen), followed by purification using the PureLink RNA mini kit (Cat#. 12183020, Invitrogen). For cell cultures, media was removed before the addition of the TRIzol reagent. Following extraction, RNA samples were treated with RNase-free DNase I (Cat#. 18068015, Invitrogen). For the first-strand synthesis of cDNA, 1–2 µg of total RNA was reverse transcribed using SensiFAST^TM cDNA Synthesis Kit (Cat#. BIO-65054, Meridian Bioscience) according to the manufacturer’s protocol. Quantitative PCR was performed using SensiMix^TM II Probe Kit (Cat#. BIO-83005) with Universal ProbeLibrary (Roche Diagnostics), and the CFX96 Touch System (Bio-Rad). Relative fold expression was normalized to β-actin (ACTB; TaqMan), hydroxymethylbilane synthase (HMBS), and hypoxanthine-guanine phosphoribosyltransferase (HPRT) housekeeping control using primer sequences listed below.