RNA isolation and reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was isolated from hOBs [2D culture in OM in the presence or absence of GlcN (200 µg/ml)] by using an RNeasy Micro kit (Qiagen GmbH) according to the manufacturer's instructions. RNA concentration and quality were measured using a NanoDrop™ ND1000 UV-VIS spectrophotometer (Isogen Life Science B.V.). cDNA was synthesized from total RNA in a 20 µl reaction volume using a High Capacity cDNA RT kit, according to the manufacturer's instructions. Finally, 100 ng cDNA was used for qPCR analysis. TaqMan Universal Master Mix II and probes for human alkaline phosphatase (ALP; assay no. Hs01029144_m1), Runx2 (assay no. Hs00231692_m1), OPN (assay no. Hs00959010_ m1), COL1A1 (assay no. Hs00164004_m1), osteocalcin (OCN; assay no. Hs01587813_g1), bone sialoprotein (BSP; assay no. Hs00913377_m1) were used according to the manufacturer's instructions. Thermocycling conditions for qPCR were as follows: Initial activation at 95°C for 10 min, followed by 40 cycles of thermal denaturation at 95°C for 15 sec and annealing/elongation at 60°C for 1 min. RPL13a (assay no. Hs04194366_g1) was used for normalization of mRNA expression. Gene expression was assessed using a CFX96TM PCR detection system (Bio-Rad Laboratories, Inc.), and relative gene expression was calculated using the comparative 2^−ΔΔCq method (21) and expressed as fold change. All reactions were performed in triplicate (n=4).