Real-time RT-PCR analysis of 5-LO and FLAP mRNA isolated from normal and osteoarthritis osteoblasts. Confluent cells were incubated for their last 48 hours in Ham's F12/DMEM medium containing 0.5% BSA. Cells were lyzed with TRIzol and RNA extracted according to the protocol described in the Materials and methods section. Plasmid DNAs containing the target gene sequences were used to generate standard curves. When comparing normal and osteoarthritis (OA) osteoblast expression levels, values were converted to numbers of molecules and the values for each sample were calculated as the ratio of the number of molecules of 5-lipoxygenase (5-LO) (a) or 5-LO-activating protein (FLAP) (b) to the number of molecules of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Values are means ± SEM for n = 4 samples for all conditions.