Production of leptin in normal and OA osteoblasts. The expression of leptin was first determined by qPCR. Confluent osteoblasts (Ob) were lized in TRIzol and RNA extracted as described in Material and methods. RNA (1 μg) was reversed transcribed followed by qPCR amplification of 100 ng cDNA using specific primers for leptin and GAPDH. The data were processed with the GeneAmp 5700 SDS software and given as threshold cycle (Ct), corresponding to the PCR cycle at which an increase in reporter fluorescence above baseline signal can first be detected. The Ct was converted to the number of molecules and the values for each sample calculated as the ratio of the number of molecules of the target gene/number of molecules of GAPDH. A) Quantification of leptin mRNA using Lep1 and Lep2 primers. Results are given as the mean value of markers relative to GAPDH ± SEM of n = 4 OA preparations. B) Quantification of leptin mRNA levels in normal and OA Ob using Lep1 primers. Results are the mean ± SEM of n = 5 normal and n = 15 OA individual Ob preparations. C) OA Ob were exposed to increasing doses of leptin and lepin mRNA levels were determined using Lep1 primers. Results are the mean ± SEM of n = 4 preparations. The protein production of leptin was next detected using a very selective ELISA. Conditioned-media of confluent normal and OA Ob incubated in HAM's F12/DMEM media containing 0.5% FBS for their last 48 hours of culture were recuperated and stored at -80°C. D) Aliquots were taken to measure leptin using a very sensitive ELISA. Results are the mean ± SEM of n = 5 normal and n = 6 OA individual Ob preparations.