Zr rna miniprep

RNA from the cell cultures was extracted using the ZR RNA mini-prep (Zymo Research Corporation, Irvine, CA, USA) according to the manufacturer’s protocol, and quantified by spectrophotometry. RNA integrity was assessed by non-denaturing agarose gel. Two hundred and twenty five nanograms of total RNA for a final concentration of 20 ng/µL was used as template for reverse transcription, performed using the High Capacity cDNA Archive kit (Thermo Fisher, Foster City, CA, USA), according to the manufacturer’s protocol.

Real-time PCR was utilized to analyze the osteogenic gene expression. The extraction procedure was performed according to [14 (link)]. Total RNA of the D1 cells was extracted using ZR RNA MiniPrep (Zymo Research, Irvine, CA, USA) after 12, 24, and 48 h of incubation in the osteoinduction medium. Total RNA (1 µg) was reverse-transcribed into cDNA using oligo-dT primers and a High Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, Waltham, MA, USA). Real-time PCR was performed using the Bio-Rad iQ5 real-time PCR detection system (Bio-Rad Laboratories Inc., Hercules, CA, USA) and SYBR green (Applied Biosystems, Waltham, MA, USA). The cDNA was amplified with BMP and osteocalcin (OC) primers using real-time PCR. The primers were as follows: BMP-2 forward, 5′-AGCTGCAAGAGACACCCTTTG-3′; BMP-2 reverse, 5′-AGCATGCCTTAGGGATTTTGGA-3′; OC forward, 5′-GAGGGCAATAAGGTAGTGAACA-3′; and OC reverse, 5′-AAGCCATACTGGTCTGATAGCTCG-3′. For the real-time PCR amplification, cDNA was denatured at 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s, 95 °C for 5 s, and 65 °C for 5 s to generate the melting curves for the confirmation of the PCR specificity. The level of relative mRNA expression was calculated from the threshold cycle and normalized with β-actin. All real-time PCR experiments were performed three times.