Rt pcr primers

Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. RT-PCR primers (synthesized by Sangon, Shanghai, China) were as follows: ALP, 5′- CAT GTT CCT GGG AGA TGG TA - 3′ and 5′- GTG TTG TAC GTC TTG GAG AGA - 3′; cbfa1, 5′- GCC GGG AAT GAT GAG AAC TA - 3′ and 5′- GGA CCG TCC ACT GTC ACT TT - 3′; COL IA1, 5′- TTA CTA CCG GGC CGA TGA - 3′ and 5′- CTG CGG ATG TTC TCA ATC TG - 3′; and GAPDH, 5′- TGT TCC TAC CCC CAA TGT ATC CG -3′ and 5′- TGC TTC ACC ACC TTC TTG ATG TCA T -3′. RT-PCR was performed using Access PCR reagent (Promega, Madison, WI) for 30 cycles of 94 °C for 30 s, 55 °C for 1.5 min, and 68 °C for 1 min, with an additional 7-min incubation at 72 °C after completion of the final cycle. A 10-μl sample of each PCR product was size-fractionated by 1.5% agarose gel electrophoresis and bands were visualized with a UV transilluminator (Tanon, Shanghai, China).
For the mineralized nodule formation assay, the mineralized matrix was analyzed using alizarin red S staining [37 (link)]. The cell cultures were rinsed with PBS and fixed in ice-cold, 90% ethanol solution for 10 min. The cells were washed with distilled water, treated with a 0.2% alizarin red S solution (Amresco, Solon, OH, USA) for 5 min, and washed with distilled water to remove the remaining staining.

According to the manufacturer’s protocol, an RNA kit was used (Qiagen, Valencia, CA, USA) to isolate total RNA from the cells. Reverse transcription reagents (Promega, Madison, WI, USA) were used to reverse transcribe RNA into complementary DNA. The RT-qPCR system (catalog no. A6002, Promega, WI, USA) was applied for qPCR according to the manufacturer’s instructions. The relative gene expression was quantified by a Rotor-Gene Q (Qiagen, Hilden, Germany) RT-qPCR system. For the RNase R assay, total RNA was incubated with or without RNase R Geneseed (3 U/mg) (Guangzhou, China) and detected by RT-qPCR. Divergent primers were designed to amplify circBBS9. The amplification products were detected by agarose gel electrophoresis and sequencing. For circRNA and mRNA, Gapdh was used as an internal control, and U6 was used as an internal control of miRNA. siRNA^circBBS9-treated and nontreated BMMs were stimulated with lipopolysaccharide and interleukin-4 and then assayed mRNA by RT-qPCR. RT PCR primers were purchased from Sangon Biotech (Shanghai, China). All primers are listed in table S1.