Platinum sybr green qpcr

BMSCs were treated with osteogenic medium (control), osteogenic medium containing 0.5 mM of DMOG, osteogenic medium containing 10 μM of Y-27632, or osteogenic medium containing 0.5 mM of DMOG and 10 μM of Y-27632 for 1, 3, and 7 days. BMSCs were harvested and the total RNA was isolated using TRIzol (Invitrogen Inc., Carlsbad, CA, USA). The 260/280 nm absorbance ratio was used to determine the purity and concentration of the RNA. The cDNA was synthesized using a SuperScript TM III First-Strand Synthesis System for RT-PCR (Invitrogen Inc., Carlsbad, CA, USA). The primer sequences were: Runx2, forward 5'-TTC AAC GAT CTG AGA TTT GTG GG-3' and reverse 5'-GGA TGA GGA ATG CGC CCT A-3'; Osterix, forward 5'-ATG GCG TCC TCT CTG CTT G-3' and reverse 5'-TGA AAG GTC AGC GTA TGG CTT-3'; and β-actin, forward 5'-GTG ACG TTG ACA TCC GTA AAG A-3' and reverse 5'-GCC GGA CTC ATC GTA CTC C-3'. Analysis was performed using an ABI prism 7000 real-time PCR system (Applied Biosystems, Foster City, CA, USA) and Platinum SYBR Green qPCR (Invitrogen Inc., Carlsbad, CA, USA). All reactions involved initial denaturation at 95°C for 2 min followed by 40 cycles of 95°C for 15 s, 54°C for 30 s, and 65°C for 20 s. The 2 -ΔΔCt method was used to represent the relative mRNA expression of target genes. β-actin was used as an internal control.