Hsa mir 214 3p

Total RNA in the cells or bones and total miRNAs in serum or exosomes were isolated using TRIzol (Life technologies) reagent and miRNeasy Serum/Plasma Kit (Qiagen) or mirVana miRNA Isolation Kit (Ambion) according to the manufacturer's protocol, respectively. After purification, the total RNA was treated with TURBO DNase (Ambion) and reverse transcribed into first-strand cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems). We used 100 ng cDNA per PCR. The TaqMan primer-probe combinations for pri-miRNAs, pre-miRNAs and miRNAs were products of Ambion. The primer for mRNAs were purchased from Shanghai Integrated Biotech Solutions Co., Ltd (Supplementary Table 6). For detection of 3′ end uridylated miR-214-3p isoforms, the following miRNA assays were used: hsa-miR-214-3p (Ambion, ID 002306), hsa-miR-214-3p-AAA (custom order: 5′-ACAGCAGGCACAGACAGGCAGUAAA-3′, hsa-miR-214-3p-UUU (custom order: 5′-ACAGCAGGCACAGACAGGCAGUUUU-3′). Real-time PCR reactions were performed using the 96-well ABI Prism 7900 HT Sequence detection system (Applied Biosystems). Please see Supplementary Table 5 for the raw data for all the real-time PCR analysis.

To investigate the functional role of selected miRNAs in regulating breast cancer biology, MDA-MB-231 cells (0.168 × 10⁶ cells/ml) were transfected with the selected miRNA precursors (pre-miR–negative control, hsa-miR-205-5p and hsa-miR-214-3p) purchased from Ambion. Cell transfection was conducted employing a reverse transfection protocol as we previously described (30 (link), 33 (link)). Briefly, pre-miRs (at 30 nM final concentration) were diluted in 50 μl of Opti-MEM (Thermo Scientific, Rockford, IL, USA), and 1.5 μl of Lipofectamine 2000 (Thermo Scientific, Rockford, IL, USA) was diluted in 50 μl Opti-MEM. The diluted pre-miRs, and Lipofectamine 2000 were then mixed and were incubated at room temperature for additional 20 min. Eight hundred microliters of transfection mixture were then transferred to the 6-well tissue culture plate and subsequently 0.168 × 10⁶ MDA-MB-231 cells/ml in transfection medium (Opti-MEM) were added to each well. After overnight incubation, the transfection cocktail was replaced with fresh DMEM without antibiotics.