Faststart sybr green master kit

Total RNA of the cells of each group was extracted using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Reverse Transcriptase (SuperScript III, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) was used for reverse transcription of total RNA to cDNA. Expression levels of the osteoblast-related genes alkaline phosphatase (ALP), osteocalcin (OCN), runt-related transcription factor 2 (RUNX2), collagen type 1 (Col-1) and bone sialoprotein (BSP) were measured using Faststart SYBR Green Master kit (Sigma-Aldrich Corp., St. Louis, MO, USA) and a real-time PCR System (LightCycler 96, Roche Diagnostics International AG, Rotkreuz, Switzerland). The expression of the genes was analyzed using the 2^−ΔΔCt method and normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene (Table 1).

Cells were treated with SAF-Hex and cultured to extract mRNA using the miRNeasy kit (Qiagen, Germany). The mRNA quantity was measured using a NanoDrop spectrophotometer (Thermo Fisher). Briefly, 1 µg of total mRNA was reverse transcribed with the Transcriptor Universal cDNA Master kit (Sigma Aldrich, US). Real-time PCR reactions were performed using the FastStart SYBR Green Master kit (Sigma Aldrich), primers, and a 50 ng cDNA template. Primer sequences were a combination of previously established [13 (link), 14 (link)] and newly designed sequences (Table S2). qPCR was performed using a Light Cycler 96 instrument (Roche Diagnostics, Germany). The thermal cycling protocol comprised an initial denaturation step at 95 °C for 10 min, followed by 45 cycles of denaturation at 95 °C for 15 s, annealing at specified temperatures for 15 s, and extension at 72 °C for 20 s. Gene expression levels were normalized using the 2^−ΔΔCt method, with GAPDH as the reference gene.