Transcription profiling by array of human MCF-7 cells with ER-beta tagged with TAP-tag at the C-term or N-term or expressing ER-alpha tagged

MCF-7 TET Off cells (MCF-7 wt) were used to produce stable clones expressing ER-beta tagged with TAP-tag respectively at the C-term and at the N-term (C-TAP-ER-beta and N-TAP-ER-beta) or expressing ER-alpha tagged (C-TAP-ER-alpha). All were grown in Dulbecco's modified Eagle's medium (DMEM). Then cells were lysed and RNA extracted were pooled. For mRNA expression profiling, 500 ng total RNA were reverse transcribed and used for synthesis of cDNA and biotinylated cRNA. Finally cRNA were hybridized for 18 hours on Illumina HumanHT-12 v3.0 BeadChips and after scanning, data analysis was performed.

本研究以MCF-7 TET关闭细胞（野生型MCF-7）为材料，构建稳定克隆：分别获得在C端（C-term）和N端（N-term）携带TAP标签（TAP-tag）的ER-beta（雌激素受体β）稳定克隆（C-TAP-ER-beta与N-TAP-ER-beta），以及携带TAP标签的ER-alpha（雌激素受体α）稳定克隆（C-TAP-ER-alpha）。所有上述细胞均培养于达尔伯克改良伊格尔培养基（Dulbecco's modified Eagle's medium, DMEM）中。随后裂解细胞，将提取得到的总RNA混合均匀。针对mRNA表达谱分析，取500 ng总RNA进行反转录，用于合成cDNA与生物素标记的cRNA。最终将cRNA在Illumina HumanHT-12 v3.0微珠芯片上进行18小时的杂交，扫描芯片后完成数据分析。