Estrogen Response and Hypoxia Regulation

As described in detail (15 (link)), oligo-dT and random primers were used to reverse transcribe RNA with qScript Flex cDNA kit (Quantabio) according to the manufacturer's instructions. To investigate whether estrogen exposure can lead to increased expression of estrogen response genes and whether cell lines grown in 5% oxygen conditions can induce carbonic anhydrase IX (CA9), which is the most well established target of HIF, quantitative RT-PCR (qRT-PCR) was performed using gene-specific primers (Table S2) and Sybr Green Master Mix (Life Technologies), and expression values were normalized relative to HPRT1 RNA expression. The 2^∧(–delta delta CT) method was used to analyze the relative changes in gene expression.