title: 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.
authors.affiliation: Faculty of Dentistry, Sir John Walsh Research Institute, University of Otago, 310 Great King Street, Dunedin, 9016, New Zealand. dawn.coates@otago.ac.nz.
authors.forename: Dawn E
authors.initials: DE
authors.lastname: Coates
authors.affiliation: Faculty of Dentistry, Sir John Walsh Research Institute, University of Otago, 310 Great King Street, Dunedin, 9016, New Zealand.
authors.forename: Sobia
authors.initials: S
authors.lastname: Zafar
authors.affiliation: Faculty of Dentistry, Sir John Walsh Research Institute, University of Otago, 310 Great King Street, Dunedin, 9016, New Zealand.
authors.forename: Trudy J
authors.initials: TJ
authors.lastname: Milne
mesh_terms: D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D019295:Computational Biology / Q000379:methods; D004164:Diphosphonates / Q000494:pharmacology; D020869:Gene Expression Profiling / Q000379:methods; D005786:Gene Expression Regulation; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D059467:Transcriptome; D000077211:Zoledronic Acid
chemical_list: D004164:Diphosphonates; D007093:Imidazoles; D000077211:Zoledronic Acid
keywords: Bisphosphonate-related osteonecrosis of the jaw (BRONJ); Geranylgeraniol; PCR arrays; Quantitative real-time reverse transcriptase PCR (qRT2-PCR); Zoledronic acid