Gene Expression Analysis by Real-Time PCR

Total RNA was extracted, and cDNA was synthesized by reverse transcription as previously described [40 (link)]. The expression of selected genes was quantified by real-time PCR using a Quant Studio7 Flex Real-Time PCR System platform (Applied Biosystems Inc, Milan, Italy). Gene-specific primers were designed using Primer Express version 2.0 software (Applied Biosystems Inc, Milan, Italy). For GPER and actin, whose genes were used as controls to obtain normalized values, the primers were 5’-ACACACCTGGGTGGACACAA-3’ (GPER forward) and 5’-GGAGCCAGAAGCCACATCTG-3’ (GPER reverse) as well as 5′-AAGCCACCCCACTTCTCTCTAA-3′ (actin forward) and 5′-CACCTCCCCTGTGTGGACTT-3′ (actin reverse), respectively. The assays were performed in triplicate and the results were normalized for actin expression and then calculated as fold induction of RNA expression.