Realplex real time pcr detection system

Total RNA was isolated using Trizol reagent (Roche Diagnostics, Indianapolis, Indiana) according to the manufacturer’s protocol. A total of 2 μg of RNA was reverse-transcribed using the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa). The SYBR green (TaKaRa) method was used with the Realplex real-time PCR detection system (Eppendorf) to detect gene expression. Real-time PCR was performed in triplicate. The sequences of the oligonucleotide primers used for real-time PCR are described in Supplementary Table 3.

MCF7 cells, MCF7-LEM4 cells, and MCF7-LEM4 cells with depletion of LEM4 expression by siRNA, grown for 3 days in phenol red-free DMEM supplemented with 5% charcoal-treated FBS. Cells were treated with either vehicle or 10 nM E2 for various time. The Simple ChIP® Enzymatic Chromatin IP Kit (Cell Signaling, catalog number: #9003) was used to perform the chromatin immunoprecipitation (ChIP) assay according to the manufacturer’s instruction. Briefly, cells were cross-linked with 1% formaldehyde for 10 min at room temperature. Glycine quenched samples were washed with ice-cold PBS, collected, and then nuclei were collected after cell lysis. Micrococcal nuclease was added to the nuclei suspension to digest the DNA for 20 min at 37 °C. Subsequently, the digest reactions were stopped by the addition of 0.5 M EDTA. Nuclear pellet were collected and were incubated in ChIP buffer with protease inhibitors for 10 min on ice. Sheared cross-linked-chromatin preparation was collected after sonication. Chromatin extracts containing DNA fragments of 150–900 base pairs were immunoprecipitated using anti-ERα or anti-IgG antibody. Quantitative real-time PCR analyses were performed using the Realplex real-time PCR detection system (Eppendorf). The sequences of the primers described in Supplementary Table 4.