2.4. Real-time quantitative polymerase chain reaction (qPCR)

Osteoblasts express different biomarker genes according to their differentiation stages. The expression of osteoblast-related genes was examined by qPCR at three different time points to identify the level of osteoblast differentiation. As described elsewhere [57], total RNA was extracted from cells using TRIzol (Invitrogen, Carlsbad, CA, USA) and a Direct-zol RNA MiniPrep kit (Zymo Research, Irvine, CA, USA) on days 5, 7, and 10. Extracted RNA was reverse transcribed into first-strand cDNA using SuperScript III Reverse Transcriptase (Invitrogen). Quantitative PCR was performed in a 20 μL volume containing 90 ng cDNA, 10 μL TaqMan Universal Master Mix II, and 1 μL TaqMan Gene Expression Assay using a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, Canoga Park, CA, USA) to quantify the expression of type I collagen, osteopontin, and osteocalcin mRNA. Graph expression was used as the endogenous control.