Myiq single color real time pcr system

Real-time quantitative reverse transcription-PCR was performed either with a Biorad MyiQ™ Single-Color Real-time PCR System (at SUNY Buffalo) or an ABI 7500 Real-time PCR System (at UTMDACC). The cDNAs were synthesized from 1.5 ìg of total RNA using SuperScript III reverse transcriptase (200 U/ìl) (Invitrogen) with random hexamers. The gene-specific primers are summarized in Table S1 in File S1. The reaction components were added as follows: 5 µl forward primer (5 µM), 5 µl reverse primer (5 µM), 12.5 µl SYBR Green (Applied Biosystems), 1 µl cDNA, Taq polymerase, and water to 25 µl. The cycling profile for each run consisted of 50°C for 3 min, template denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 15 s, a primer annealing-elongation step at 60°C for 30 s using the default ramp rate. This was followed by a final step of 40°C for 1 min. All reactions were run in triplicate and the mean values were used in the analysis. The cycle threshold (C_t) values were determined using the instrument software. The change in gene expression levels was determined by normalizing mRNA levels of the gene of interest to the mRNA level of the housekeeping gene glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) using the comparative C_t method.

Total cellular RNA was isolated from cells cultured with different concentrations of quercetin for 1, 3, 6, 12 and 24 hours, as previously described. At each time point, the cells were washed twice with PBS, and RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The RNA was first separated into an aqueous phase by adding chloroform and was then precipitated with isopropanol. The RNA precipitate was rinsed with 70% ethanol and treated with the RNase inhibitor diethyl pyrocarbonate (DEPC, Sigma) and was finally solubilized in sterile DEPC water. Complementary DNA (cDNA) was then synthesized using a Prime-Script RT reagent kit (Takara Bio, Japan) according to manufacturer’s recommendations. Highly purified gene-specific primers for Runx2, COL1, BSP, BMP-2, OPN, OCN, VEGF, angiogenin-1 (ANG-1) and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were commercially synthesized (Shengong, China). Quantification of the cDNA of bone marker genes was performed with a Bio-Rad My^iQ single-color real-time PCR system. All experiments were performed in triplicate.

To measure the expression of osteogenic and angiogenic genes of rBMSCs seeded on different materials as previously described, the RT-PCR analysis was performed at 4, 7, and 10 days. At each time point, after collecting the cells, the RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, United States), and complementary DNA (cDNA) was then synthesized using a Prime-Script RT reagent kit (Takara Bio, Japan) following the manufacturer’s recommendations. Quantification for ALP, bone morphogenetic protein 2 (BMP-2), osteopontin (OPN), vascular endothelial growth factor (VEGF), and angiogenin-1 (ANG-1) were analyzed with a Bio-Rad MyiQ single-color real-time PCR system, while glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control for normalization. All experiments were performed in triplicate.