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Figure 1. Quantitation of OPN and OC expression levels by real-time PCR. Relative levels of OPN (A,B,C) and OC (D,E,F) mRNA were measured by real-time PCR and standardized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels, which were used as an internal control. The primer sequences for quantitative PCR are as follows: 5'-CCCTGGCTGCGCTCTGT-3' and 5'-GCGCCGGAGTCTGTTCAC-3' for OC, and 5'-ACTTTCACTCCAATCGTCCCTACA-3' and 5'-GGCATCAGGATACTGTTCATCAGA-3' for OPN. Quantification of GAPDH mRNA (internal control) was performed with TaqMan® Rodent GAPDH Control Reagents (VICTM Probe) (Applied Biosystems, Foster City, CA, USA). Detection of OPN and OC mRNAs was performed with the use of TaqMan® FAM-MGB probes with the following sequences: 5'-FAM-CTGACAAAGCCTTCATGTC-MGB-3' for OC and 5'-FAM-TCAAAGTCTAGGAGTTTCC-MGB-3' for OPN. Quantitative PCR analysis was performed with the use of an ABI Prism 7000 Sequence Detection System and TaqMan® Universal PCR Master Mix (Applied Biosystems) for 40 cycles of 95°C for 15 sec and 60°C for 1 min as described in the manufacturer’s protocol. We calculated cellular mRNA levels as relative values, dividing each mRNA level by the GAPDH mRNA level of each sample as internal control. Relative mRNA levels—without treatment (open circles), following treatment with 0.1 mM (C and F), 0.5 mM (B and E), and 1 mM (A and D) Na-PO4 buffer (closed triangles), and following treatment with poly(P) (closed squares)—are shown. Error bars represent the mean ± SD of three independent analyses.
