Quantitative Real-Time Gene Profiling of Human Alveolar Osteoblasts Using a One-Step System

The use of quantitative real-time reverse transcriptase PCR (qRT²-PCR) for the identification of differentially regulated genes is a powerful technology. The protocol presented here uses qRT²-PCR gene arrays to investigate the regulation of 84 angiogenic related genes in human primary alveolar osteoblasts following treatment with the bisphosphonate, zoledronic acid, and geranylgeraniol (GGOH). GGOH has potential as a therapeutic agent for medication-related osteonecrosis of the jaw, a serious side effect resulting from treatment for metastatic cancer (Zafar S, Coates DE, Cullinan MP, Drummond BK, Milne T, Seymour GJ. J Oral Pathol Med 43:711-721, 2014). The isolation of the primary osteoblast cells follows the methods described by Dillon et al. (Method Mol Biol 816:3-18, 2012) with a new RNA extraction technique described fully. The method highlights the importance of obtaining high-quality RNA which is DNA-free. Relative levels of gene expression are normalized against selected reference genes (HKG) and a number of examples of how fold regulation (2^-ΔΔCq) and gene expression level (2^-ΔCq) data can be presented are given.