Abstract
RNAi screening technology has revealed unknown determinants of various biological signaling pathways in biomedical studies. This protocol provided detailed information about how to use RNAi screening to identify proliferation determinants in breast tumor cells. siRNA-based libraries targeting against Estrogen receptor (ER)-network, including 631 genes relevant to estrogen signaling, was constructed for screening in breast cancer cells. Briefly, reverse transfection of siRNA induced transient gene knockdown in MCF7 cells. First, the transfection reagent for MCF7 cells was selected. Next, the Z'-score assay was used to monitor if screening conditions yielded efficiently. Then, the ER-network siRNA library screening was preceded by automatic machines under optimized experimental conditions.