Osteoblast Gene Expression Analysis

Osteoblasts cells were seeded in 6-well plates at a density of 1 × 10⁶ cells/well. After 6-day culture, cells were treated with the TPF at concentrations of 0, 50, and 100 ng/ml for 48 h. Total RNA from the cells of each well was isolated respectively using NucleoSpin (Macherey–Nagel, Duren, Germany). RNA aliquots were reverse transcribed to complementary DNAs by using an oligo (dT) primer (Roche), deoxynucleotide triphosphate (dNTP), and Moloney murine leukemia virus (M-MuLV) reverse transcriptase (Fermentas, Hanover, MD). The complementary DNA products were subjected to PCR amplification with gene-specific primers for mouse Alp, Osteocalcin and Type 1 collagen [23 (link)]. Real-time RT-PCR amplification was performed using a Light Cycler System (Roche) with a Platinum SYBR Green qPCR Super Mix UDG kit (Invitrogen, Carlsbad, CA).