Optimised real-time quantitative PCR assays for RANKL regulated genes.
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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.
Molecular and cellular probes.
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