RT-qPCR

Total RNA was reverse transcribed to cDNA by a Reverse Transcription kit (Roche Diagnostics, Basel Switzerland). RT-qPCR was performed using an Applied Biosystems 7900HT thermal cycler, with a 20 µl PCR reaction mixture containing 10 µl of 2X LightCycler 480 SYBR Green I Master mix (Roche Diagnostics). The following primers were used: BAI-1, forward 5′-CCGCTGTGTTTCCATTGACTA-3′ and reverse 5′-ACCACAAACACGGATGCTTCA-3′; glyceraldehyde 3-phosphate dehydrogenase (GAPDH), forward 5′-GAAGGTCGGAGTCAACGGAT-3′ and reverse 5′-CTGGAAGATGGTGATGGGATT-3′. The qPCR cycling conditions were as follows: 95°C for 10 min followed by 40 cycles of 95°C for 10 sec, 58°C for 20 sec and 72°C for 20 sec, followed by 72°C for 5 min. qPCR was used to measure the gene expression levels of BAI-1. The results were normalized using the 2^−ΔΔCq method (15).