Hs00959010 m1

Total RNA was extracted according to the previous protocol with minor modifications^{27 (link), 28 (link)}. Real-time PCR was performed following manufacturer’s instructions using an ABI Prism 7700 with SYBR green I as a double-stranded DNA-specific dye (PE-Applied Biosystems, Cheshire, UK). Primers of IL-8, TNF-α, MCP-1, and hMMP7 were listed in Table 1 and were constructed to be compatible with a single RT-PCR thermal profile (95 °C for 10 min, 40 cycles at 95 °C for 30 s, and 60 °C for 1 min). The accumulation of the PCR product was recorded in real time (PE-Applied Biosystems). In addition, the TaqMan gene expression assays were used to measure GRO-α (Hs00236937_m1, Applied Biosystems) and SPP1 (Hs00959010_m1, Applied Biosystems) mRNA expression. The results of the mRNA levels for the different genes are displayed as transcript levels of the analyzed genes relative to GAPDH. The statistical analyses were performed with Student’s t test. Statistically significant were considered as p-values of ≤0.05.

Total RNA was extracted from PBMCs with the NucleoSpin RNA kit (Macherey&Nagel, Germany) in accordance with the instructions of the supplier. Total RNA was eluted in a 50-µl volume of RNase-free water. RNA concentration was analyzed by NanoDrop spectrophotometer (ND-1000 Spectrophotometer, NanoDrop Technologies, Inc, USA). 0.1 μg of total RNA was reverse transcribed into cDNA using a commercially available High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA) according to the manufacturer’s instruction. Detection of mRNA level in the samples was performed using real-time PCR (RT-PCR) on ABI Prism 7500 Sequence Detector (Applied Biosystems, USA). The analysis of OPN gene expression was performed using a human commercial available assay Hs00959010_m1 (Applied Biosystems, USA). Fluorescence intensities were analyzed using the manufacturer’s software (7500 Software v2.05) and relative amounts were obtained using the 2−∆∆Ct method and normalized for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH).