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Total RNA was resuspended in 12 |xl of DEPC-treated ddH2O. Reverse transcriptase polymerase chain reaction (RT-PCR) amplifications were performed using primers designed on the basis of the mouse Sox9 as previously reported. The sense primer was Sox9-1, 5'-GTG GCA AGT ATT GGT CAA-3'; the antisense primer was Sox9-2, 5'-GAA CAG ACT CAC ATC TCT-3'. For expression control, amplification of oligonucleotides used as internal controls were Actin 1 and Actin 2 as previously described. The amplification reactions were carried out by means of the One Step RT-PCR kit (Life Technologies Invitrogen) using 3 |xl of total RNA per reaction, 1X buffer reaction mix containing 0.2 mM of each dNTP, 1.2 mM MgSO4, and 200 ng of p-actin primers or 300 ng of Sox9 primers in separate tubes. The enzyme used was a mixture of Superscript II RT and Taq DNA polymerase Platinum (Life Technologies Invitrogen) in a volume of 20|xl. The amplification program was 50°C for 30 min (once); 94°C/5 min (once), 94°C/30 sec, 56°C/60 sec, 72°C/60 sec (25 cycles), 72°C/10 min (once) within the linear range previously described. In order to confirm that the amplified products were not genomic DNA in origin, we used an RT-free control (Taq DNA polymerase but not template DNA added) for each RT-PCR.