Real-time quantitative PCR analysis of Vegf knockdown on expression of extracellular matrix and adhension molecules in male and female primary murine osteoblasts

Primary murine osteoblasts, isolated from the long bones of 4-day old male and female Vegffl/fl mice, were cultured prior to transduction with adenovirus-Cre-GFP at an multiplicity of infection of 100, to knockdown Vegf expression (OBVEGFKO) in vitro for six days. Control cells were transduced with an adenovirus expressing GFP (WT). Following this, cells were stepped down in low serum media containing 1% FBS for 24 hours, after which the conditioned media was collected for knockdown validation using an VEGF ELISA. We used the Qiagen Extracellular Matrix and Adhension Molecule RT2 Profiler PCR Array to assay the sexually dimorphic effects of Vegf on the expression of genes associated with bone matrix formation and mineralisation, remodelling and angiogenesis. qPCR gene expression array. Osteoblasts isolated from 4-day old male and female Vegffl/fl mice were cultured prior to being transfected with adenovirus expressing Cre-recombinase-GFP to knockdown Vegf expression or adenovirus expressing GFP alone. Knockdown was validated using an VEGF ELISA. Equal amount total RNA (500 ng) was purified from each treatment group before reverse transcription and subsequent expression profiling.