Quantitative Real-Time Gene Profiling of Human Alveolar Osteoblasts

Abstract

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 (ZA), and geranylgeraniol (GGOH). GGOH has potential as a therapeutic agent for Bisphosphate-Related Osteonecrosis of the Jaw (BRONJ), a serious side-effect resulting from the treatment for metastatic cancer (Zafar et al., J Oral Pathol Med 43:711–721, 2014; Ruggiero, Ann NY Acad Sci 1218:38–46, 2011). The isolation of the primary osteoblast cells follows the methods previously described (Dillon et al., Methods 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 housekeeping genes (HKG) and a number of examples of how fold regulation (2^−∆∆Cq) and gene expression level (2^−∆Cq) data can be presented are given.