Qmmuced0041364

The osteogenic biomarkers including Runt related transcription factor 2 (Runx2), osteocalcin (OCN), and bone sialoprotein (BSP) were analyzed on day 7 using real-time PCR. Quantitative gene expression analysis was carried out according to the product instructions. Briefly, total cellular RNA was extracted using a miRNeasy Mini Kit (Qiagen, Valencia, CA). The concentration of total RNA was measured using a NanoDrop™ One spectrophotometer (Thermo Scientific, Pittsburgh, PA). Equivalent amount of RNA was processed to generate a complementary strand of DNA (cDNA) using an iScript™ cDNA Synthesis Kit (Bio-Rad Hercules, CA). Runx2, OCN, and BSP were expressed on a CFX Connect™ (Bio-Rad Hercules, CA) utilizing the iQ SYBR® Green Supermix Kit (Bio-Rad Hercules, CA). Gene primers of Runx2 (qMmuCED0049270) and OCN (qMmuCED0041364) were purchased from Bio-Rad (Hercules, CA), while the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and BSP were synthesized at the University of Iowa (Table S1).

Calcium deposition on the cell-scaffold constructs was evaluated by a total calcium LiquiColor® kit (Stanbio Laboratory, TX) according to our previous research work (Yao, Fuglsby, Zheng, & Sun, 2020 (link)) after 3 weeks of cell culture. The calcium content was calculated from the following Equation (4):
$$\text{ Calcium }(\text{mg}/\text{dL})=\left(A_u/A_s\right)\times 10$$
where A_u and A_s are the absorbance values of sample and standard at 550 nm, respectively. 2.5.4. RNA extraction and quantitative real-time PCR.
The osteogenic biomarkers including Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and bone sialoprotein (BSP) were analyzed on varied periods using real-time PCR according to our previous report (Hu et al., 2020 (link)). Gene primers of RUNX2 (qMmuCED0049270) and OCN (qMmuCED0041364) were purchased from Bio-Rad, while the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and BSP were synthesized at the University of Iowa (Table S1).