2.2.6. RT-qPCR Detection of Transcription Levels of Osteogenic Differentiation Markers

Using 1,25-dihydroxyvitamin D₃ to intervene and culture SCAPs to day 7 and day 14, then use the total RNA extraction kit to extract total RNA in the sample. Detect RNA concentration with a NanoDrop spectrophotometer. According to the RNA concentration, the template cDNA was obtained using a reverse transcription kit and diluted 10 times with DEPC water. Use SYBR green as a fluorescent dye, and use a real-time fluorescence quantitative PCR instrument to perform PCR amplification reaction in a 10 μL reaction system. The amplification procedure was as follows: 95°C for 10 min, 95°C for 10 min, 55°C for 15 s, and 72°C extends for 15 s for a total of 40 cycles. The housekeeping gene GAPDH is adopted as an internal reference, normalizing the detection results by calculating the relative quantitative ΔΔCT fluorescence. Table 1 illustrates the sequences of the osteogenic-related genes detected in the experiment.

Illustration of the sequences of the osteogenic-related genes, detected in the experiment. Primer sequence list.