A total of three genes related to bone metabolism and mineralization were included in the present study: Runx2 (ID: 860), SP7 (ID: 121340), and ALPL (ID: 249). One-step real-time quantitative polymerase chain reaction (RT-qPCR) was performed by using the Reliance Multiplex RT-qPCR Supermix combined with the following Prime PCR probe assays (BioRad, Hercules, CA, USA): Runx2 (qHsaCEP0051329), SP7 (qHsaCEP0025867), and ALPL (qHsaCEP0053252). As suggested by Abuna et al. [56 (link)], Eif2b1 (qHsaCIP0030434) and YWHAZ (qHsaCIP0029093) were selected as reference genes, considering a wide range of algorithms for assessing expression stability in osteoblasts. Assays were conducted in a CFX96 Touch Real-Time PCR Detection System (BioRad, Hercules, CA, USA), and the thermal cycling conditions were 50 °C (10 min) for reverse transcription, 95 °C (10 min) for DNA polymerase activation and template denaturation, and 40 cycles at 95 °C (10 s) and 60 °C (30 s) in a 10 µL reaction volume, according to manufacturer’s recommendations. For each biological sample, a total of three technical replicates were included. Multiplexing was validated experimentally after comparing the Cq values and amplification efficiencies of multiplex and singleplex reactions. For RT-qPCR efficiency calculation, five 5-fold serial dilution assays were performed, and the standard curve method was applied [57 (link)].
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