RT-PCR.

Pellets were digested with 3 mg/ml collagenase, 1 mg/ml hyaluronidase and 0.25% trypsin for about 3 h at 37C. Total RNA was extracted by using RNAqueous kit (Ambion, TX). RNA was converted to cDNA and amplified by the Titan One Tube RT-PCR System (Roche Molecular Biochemicals, Germany), according to the manufacturers recommendations. RT was performed by a 30 min incubation at 50C, followed by a 2-min incubation at 94C to inac-tivate the reverse transcriptase. PCR amplification conditions for the resulting cDNAs was performed by 35 cycles of 94C for 30 s, 58C for 45 s, and 68C for 45 s, in which 68C the step was increased by 5 s every cycle after 10 cycles. The reaction products were resolved by electrophoresis on a 2% agarose gel and visualized with ethidium bromide. PCR primers were as follows. BMP-6 Enhances Chondrogenesis in a Subpopulation of Human Marrow Stromal Cells. 2001