MDA-MB-453 and MDA-MB-231 cells were seeded into 6-well plates at a density of 1×106 cells/well and transfection was performed when ~90% confluence was reached following manufacturer's protocol. Short hairpin RNA (shRNA) targeted against TTN-AS1 (shRNA-TTN-AS1-1 or shRNA-TTN-AS1-2) and a scrambled shRNA that served as the negative control (shRNA-NC), were designed and synthesized by Shanghai GenePharma Co., Ltd. The shRNAs (3 µg) were transfected into the TNBC cells using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. In addition, microRNA (miR/miRNA)-211-5p mimic, miR-211-5p inhibitor and their corresponding negative control (miR-NC) obtained from Shanghai GenePharma Co., Ltd., were transfected into the cells at a concentration of 40 nM using Lipofectamine® 3000 as previously described (15 (link)). Cells were collected 24 h following transfection, and transfection efficiency was evaluated by reverse transcription-quantitative PCR (RT-qPCR).
Protocol detail
Silencing TTN-AS1 and miR-211-5p in TNBC
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Corresponding Organization :
Other organizations : Nanjing Maternity and Child Health Care Hospital, Nanjing Medical University
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Variable analysis
independent variables
- ShRNA targeted against TTN-AS1 (shRNA-TTN-AS1-1 or shRNA-TTN-AS1-2)
- Scrambled shRNA that served as the negative control (shRNA-NC)
- MiR-211-5p mimic
- MiR-211-5p inhibitor
- Corresponding negative control (miR-NC)
- Transfection efficiency evaluated by reverse transcription-quantitative PCR (RT-qPCR)
- Cell density (1×10^6 cells/well)
- Cell confluence (~90% when transfection was performed)
- Scrambled shRNA (shRNA-NC) as a negative control
- Corresponding negative control (miR-NC) for miR-211-5p mimic and inhibitor
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