Quantitative Gene Expression Analysis of Fracture Callus

The callus was extracted from 46 fractured femurs by cutting the bone 2 mm away from each edge of the callus. An equal length of the diaphysis was extracted from the contralateral limbs. The bone samples were then flash-frozen in liquid nitrogen and processed using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. RNA was purified using Qiaquick RNeasy mini-kit (Qiagen, Valencia, CA) and cDNA was generated from total RNA using the QuantiTect Reverse Transcription Kit (Qiagen). Quantitative reverse transcription PCR (qPCR) was performed using primer and TaqMan probe sets (Applied Biosystems, Foster City, CA) and QuantiFast Probe PCR kit (Qiagen) on a Mastercycler realplex2 (Eppendorf, Westbury, NY). Amplification conditions were 95°C for 3 min, followed by 40 cycles at 95°C for 3 s and 60°C for 30 s. Quantitative PCR results were first normalized to ActB (Mm00607939_s1) transcript to yield ΔC_t, and then normalized to contralateral limb to yield ΔΔC_t. Results are expressed as 2^−ΔΔCt .^{34 (link)} Genes probed were collagen Type 1 alpha 1 (Col1a1, Mm00801666_g1), collagen Type 2 alpha 1 (Col2a1, Mm01309565_m1), collagen Type 10 alpha 1 (Col10a1, Mm00487041_m1), SDF-1 (Sdf1, Mm00445553_m1), bone sialoprotein (Ibsp, Mm00492555_m1), vascular endothelial growth factor (Vegf, Mm01281449_m1), Annexin A5 (AnxA5, Mm01293059_m1), early growth response 1 (Egr1, Mm00656724_m1), nitric oxide synthase 2 (Nos2, Mm00440502_m1), and mechanistic target of rapamycin (mTOR; Frap1, Mm00444968_m1).