Chloroform isoamyl alcohol 24 1

To analyze the gene expression of common osteoblast markers, hMSCs and oMSCs were induced towards the osteoblast lineage, and real-time polymerase chain reaction (RT-PCR) was performed, as described previously [22 (link)]. Briefly, TRIzol reagent (Ambion, Life Technologies, Darmstadt, Germany) and chloroform:isoamyl alcohol (24:1) (PanReac AppliChem, Darmstadt, Germany) were used for mRNA extraction. Then, 1 µg mRNA was reverse transcribed using a Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany), and RT-PCR was conducted using LightCycler 480 SYBR Green I Master according to the manufacturer’s instructions (Roche Diagnostics GmbH). Amplifications ran at 95 °C for denaturation, 60 °C for primer annealing, and 72 °C for primer extension 10 s each for 45 cycles. Primer sequences are listed in Table 1. Data analysis was performed using the ddCT method [60 (link)] determined by normalization to GAPDH [44 (link)].

To analyze the gene expression of common osteoblast markers, primary MSCs and the cell line MG63 were induced toward the osteoblast lineage after repeated and single SWA sessions, and real-time polymerase chain reaction (RT-PCR) was performed as described previously (Haddouti et al., 2020a (link)). In brief, TRIzol reagent (Ambion, Life Technologies, Darmstadt, Germany) and chloroform: isoamyl alcohol (24:1) (PanReac AppliChem, Darmstadt, Germany) were used for mRNA extraction. Then, 1 µg mRNA was reverse transcribed using a Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany), and RT-PCR was conducted using LightCycler 480 SYBR Green I Master According to the manufacturer’s instructions (Roche Diagnostics GmbH). Amplifications ran at 95°C for denaturation, 60°C for primer annealing, and 72°C for primer extension for 10 s each for 45 cycles. Primer sequences are listed in Table 2. Data analysis was performed using the ddCT method (Livak and Schmittgen, 2001 (link)) determined by normalization to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Wiraja et al., 2016 (link)).