2.6. Real-time PCR analysis for osteoblast differentiation markers

The hPDL cells were treated with and without DMP1 protein for 72 h. Total RNA from hPDL cells was extracted using Isol-RNA Lysis Reagent (Gene All Biotechnology, Korea), and 1 μg/ml of RNA per sample was converted to cDNA using a reverse transcriptase kit (Promega, Madison, WI, USA). Quantitative RT-PCR was performed using a Lightcycler Nano real-time polymerase chain reaction machine (Roche Applied Science, Indianapolis, IN, USA) using Fast Start Essential DNA Green Master (Roche Applied Science). Primers specific to ALP, BMP2, CBFA1, COL1, OPN, OSX and WNT3a were used for this protocol, and the RT-PCR conditions were set as follows: denaturation at 94 °C for 10 min, annealing at 60 °C for 10 s, and extension at 72 °C for 10 s for 45 cycles. GAPDH was used as a reference gene for the internal control. The primer sequences are shown in Table 1. The data were statistically analysed by two-way ANOVA.

Primer sequence for gene expression analysis using RT-PCR.