Rt2 real time sybr green reagents

Total RNA was isolated from chondrocyte cultures with TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. The RNA was reverse transcribed with qScript cDNA Supermix reagents (Quanta BioSciences, Gaithersburg, MD) and amplified at 42 °C for 30 minutes. For real-time reverse transcription–polymerase chain reaction (RT-PCR), the PCR products were detected using RT² Real-Time SYBR Green reagents (SABiosciences, Frederick, MD). Primer-specific amplification was performed at 60 °C for 30 seconds. However, fluorescence quantification was performed at a higher temperature (72°C). The primers pair sequences for bovine genes, forward and reverse, are as follows; GAPDH, 5′-ATTCTGGCAAAGTGGACATCGTCG-3′, 5′-ATGGCC TTTCCATTGATGACGAGC-3′; ADAMTS4, 5′-TCACTG ACTTCCTAGACAATGG-3′, 5′-ACTGGCGGTCAGCGT CGTAGT-3′; ADAMTS5, 5′-CACCGTGGCTCACGAAA TTG-3′, 5′-GGAGCCGAAATTTTCTTCACAGA-3′. All primers were obtained from Integrated DNA Technologies (Coralville, IA). Thermal cycling and fluorescence detection were performed using the SmartCycler System (Cepheid). Real-time PCR efficiencies and the fold increase in copy numbers of messenger RNA (mRNA) were calculated as described previously.^{34 (link)} The data are presented as mean ± standard deviation (SD) by setting negative control as 1.0, and analyzed by Student’s t test. P values <0.05 were considered significant.