U6 primer

Total RNA was isolated from cultured cells using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Real-time RT-PCR-based detection of mature miR-675 and U6 snRNA was achieved with the miRNA Detection kit (including a universe primer, U6 primers, Qiagen) and miR675 specific upsteam primers (Origene, USA). qRT-PCR was performed with a StepOnePlus real-time PCR system (Applied Biosystems), a SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) and Power SYBR Green PCR Master Mix (Applied Biosystems) in accordance with the manufacturers' protocols. Each sample was run in triplicate. C_t values for miR675 were calculated and normalized to C_t values for U6 snRNA. The following primers were used: human mi675P1: 5′-TGGTGCGGAGAGGGC-3′ and P2:5′-GAACATGTCTGCGTATCTC-3′. U6 primer:P1:5′-GCTTCGGCAGCACATATACT-3′;P2:5′-GGAACGCTTCACGAATTTGC-3′

Total RNA was isolated from cells using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Real-time RT-PCR-based detection of mature miR-15a and U6 snRNA was achieved with the miRNA Detection kit (including a universe primer, U6 primers, Qiagen) and miR15a specific upsteam primers (Origene, USA).

MCF-7-vector and MCF-7-miR-155 cells were harvested for total RNA extraction using Qiagen RNeasy RNA purification system or for microRNA miRNeasy purification system per manufacturer’s protocol (Qiagen, Valencia, CA). Quantity and quality of the RNA and miRNA were determined by absorbance at 260 and 280 nm using the NanoDrop ND-1000. 2 ug of total RNA was reverse-transcribed using the iScript kit (BioRad Laboratories, Hercules, CA) and qPCR was performed using SYBR-green (Bio-Rad Laboratories, Hercules, CA). β-Actin, PgR, ERα, BCL-2, SDF-1, SERPIN3A, Rictor, TSC1, Raptor, Deptor, p70s6 kinase, and Rheb genes were amplified n > 3. E2 stimulation experiments, cells were grown in 5% DMEM for 48 hours prior to treatment with 1 nM E2 or DMSO for 24 hours. RAD001 in vitro experiments cells were pre-treated for 30 minutes with 20 nM RAD001 followed by 100 pM E2 or DMSO. miRNA was reverse–transcribed using the SABiosciences RT² miRNA first strand kit (Qiagen, Valencia, CA) and qPCR was performed using SABiosciences SYBR green, miR-155 primer, U6 primer, and SA- Bioscience RT² cancer miRNA array plate (MAH-102A) were purchased from Qiagen (Valencia, CA). Data was analyzed by comparing relative target gene expression to β-actin for mRNA and U6 for miRNA. Relative gene expression was analyzed using 2-ΔΔCt method [44 (link)].

Real Time RT-PCR MDA-MB-231-vector and MDA-MB-miR-200b cells were harvested for total RNA extraction using Qiagen RNeasy RNA purification system or for microRNA miRNeasy purification system per manufacturer's protocol (Qiagen, Valencia, CA). Quantity and quality of the RNA and miRNA were determined by absorbance at 260 and 280 nm using the NanoDrop ND-1000. 2ug of total RNA was reverse-transcribed using the iScript kit (BioRad Laboratories, Hercules, CA) and qPCR was performed using SYBR-green (Bio-Rad Laboratories, Hercules, CA). β-Actin, CDH1, PRKCA, ZEB1, and ZEB2 amplified n > 3. miRNA was reverse–transcribed using the SABiosciences RT2 miRNA first strand kit (Qiagen, Valencia, CA) and qPCR was performed using SABiosciences SYBR green, miR-200b-3p, miR-200b-5p, and U6 primer purchased from Qiagen (Valencia, CA). Data was analyzed by comparing relative target gene expression to β-actin for mRNA and U6 for miRNA. Relative gene expression was analyzed using 2-ΔΔCt method [57 (link)].