Quantitative RT-PCR Analysis of Estrogen Receptors

Reverse transcription and quantitative real-time-PCR were performed as described previously [34 (link),35 (link)]. Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and reverse transcribed using the RevertAid H Minus cDNA Synthesis Kit (ThermoFisher Scientific, Darmstadt, Germany). Reactions where RNA or reverse transcriptase had been omitted were used as negative controls. PCR products were obtained using gene-specific primers reported previously [36 (link)] and SYBR Green (Applied Biosystems, Darmstadt, Germany) in a 7000 ABI Prism Instrument (Applied Biosystems, Darmstadt, Germany). The levels of the target genes were normalized to beta actin (Actb) housekeeping mRNA levels. The primer sequences were the following: Esr1 forward 5’-CAGGACCAGCCCGATTCC-3’ and reverse 5’-TTAGGGTACATGGGTGAGAGTTTG-3’; Esr2 forward 5’-CGCTCGGCATGGACAAC-3’ and reverse 5’-CCCATGCGGTGGAGAGTAAT-3’; Actb forward 5’-TGCCCCTCGTGCTGTTTT-3’ and reverse 5’-TCTGTCCCATGCCAACCAT-3’.