Gene Expression Profiling by Real-Time PCR

Total RNA was extracted, and cDNA was synthesized by reverse transcription as previously described [31 (link)]. The expression of selected genes was quantified by real-time PCR using platform Quant Studio7 Flex Real-Time PCR System (Thermo Fisher Scientific). Gene-specific primers were designed using Primer Express version 2.0 software (Applied Biosystems) and are as follows: 5′-ACCTATGACCTGCTTGGTGC-3′ (HIF-1α forward) and 5′-GGCTGTGTCGACTGAGGAAA-3′ (HIF-1α reverse); 5′-TTAAAGCCCGCCTGACAGA-3′ (IL-1β forward) and 5′-GCGAATGACAGAGGGTTTCTTAG-3′ (IL-1β reverse); 5′-TTACAGAGGGAAAACGACACCT-3′ (GPER forward) and 5′-GTGGGTCTTCCTCAGAAGGG-3′ (GPER reverse); 5′-AGTCCCTGAGCATCTACGGT-3′ (COX2 forward) and 5′-CATCATCAGACCAGGCACCA-3′ (COX2 reverse); 5′-AAGCCACCCCACTTCTCTCTAA-3′ (ACTB forward) and 5′-CACCTCCCCTGTGTGGACTT-3′ (ACTB reverse). Assays were performed in triplicate and the results were normalized for actin beta (ACTB) expression and then calculated as fold induction of RNA expression.
PCR arrays were performed using a TaqMan™ Human Tumor Metastasis Array (Thermo Fisher Scientific) according to the manufacturer’s instructions. The amplification reaction and the results analysis were carried out using platform Quant Studio7 Flex Real-Time PCR System (Thermo Fisher Scientific).