title: Optimised real-time quantitative PCR assays for RANKL regulated genes.
abstract: Osteoclasts are multinucleated giant cells that differentiate from precursors of the monocyte-macrophage lineage. We used receptor activator of NF-kappa B ligand (RANKL) and macrophage colony stimulating factor (M-CSF) to differentiate authentic human osteoclasts from peripheral blood mononuclear cells (PBMCs). We previously described a series of genes that are strongly regulated by RANKL. Here, we provide a set of reliable quantitative real-time PCR based assays of RANKL regulated genes as reference genes that may prove useful in the study of human osteoclasts. The SYBR-green I assays are free of primer dimer and other artefacts, and are designed to amplify in parallel, thus permitting simultaneous analysis of 12 genes. Optimised primers for 18S rRNA provide a valid housekeeping reference gene. Standard curves have been constructed for all assays, thus allowing for absolute quantification of mRNA transcript copy number. As an example, the regulation of expression of the chemokine RANTES in osteoclasts is demonstrated. These gene assays have potential utility in a variety of cell types, tissues and organs, in addition to macrophages and osteoclasts.
authors.affiliation: School of Health Science, Griffith University, Gold Coast Campus, Parklands Drive, Southport, Qld 4215, Australia. r.granfar@student.griffith.edu.au
authors.forename: Rouha M S
authors.initials: RM
authors.lastname: Granfar
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Day
authors.forename: Michael S
authors.initials: MS
authors.lastname: Kim
authors.forename: Nigel A
authors.initials: NA
authors.lastname: Morrison
mesh_terms: D052160:Benzothiazoles; D002352:Carrier Proteins / Q000502:physiology*; D002454:Cell Differentiation; D002478:Cells, Cultured; D018946:Chemokine CCL5 / Q000096:biosynthesis / Q000235:genetics; D017931:DNA Primers; D003959:Diamines; D005456:Fluorescent Dyes; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000502:physiology*; D009930:Organic Chemicals; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011804:Quinolines; D053245:RANK Ligand; D012333:RNA, Messenger / Q000032:analysis; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D053246:Receptor Activator of Nuclear Factor-kappa B; D012016:Reference Values
chemical_list: D052160:Benzothiazoles; D002352:Carrier Proteins; D018946:Chemokine CCL5; D017931:DNA Primers; D003959:Diamines; D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D009930:Organic Chemicals; D011804:Quinolines; D053245:RANK Ligand; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S; D053246:Receptor Activator of Nuclear Factor-kappa B; C505686:TNFRSF11A protein, human; C505688:TNFSF11 protein, human; C098022:SYBR Green I