Three human NSCLC cell lines: H460, A-549 and H1299 were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA) and were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco; Thermo Fisher Scientific, Inc., Grand Island, NY, USA) containing 10% fetal bovine serum and penicillin-streptomycin at 37°C under a humidified atmosphere of 5% CO2. Each of the in vitro experiments was performed 3 times. Before transfection, LUAD cells were plated in 96-well plates at 2.5×103 cells/well and maintained at 37°C for 24 h. Blank control, negative mimic control, miR-183-5p mimic, negative inhibitor control and miR-183-5p inhibitor (Ambion; Thermo Fisher Scientific, Inc., Carlsbad, CA, USA) were transfected in LUAD cell lines at a final concentration of 60 nmol/l with Lipofectamine 2000 following the manufacturer's instructions. The concentration for transfections was determined based on previous studies (21 (link),22 (link)). miR-183-5p expression was detected with qPCR in Applied Biosystems PCR 7900 system as stated previously (23 (link)–26 (link)).
Protocol detail
LUAD Cell Line Transfection with miR-183-5p
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Atmosphere
Cell lines
Cells
Cultured medium
Fetal bovine serum
Human
Lipofectamine 2000
Nsclc
Penicillin
Streptomycin
Transfections
Corresponding Organization :
Other organizations : First Affiliated Hospital of GuangXi Medical University, Guangxi Medical University
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Variable analysis
independent variables
- MiR-183-5p mimic
- MiR-183-5p inhibitor
- MiR-183-5p expression
- Cell lines (H460, A-549, H1299)
- Culture medium (DMEM with 10% FBS and penicillin-streptomycin)
- Incubation conditions (37°C, 5% CO2)
- Blank control
- Negative mimic control
- Negative inhibitor control
- The independent variables are the transfection of miR-183-5p mimic and miR-183-5p inhibitor. The dependent variable is the expression of miR-183-5p, which was measured using qPCR. The control variables include the cell lines, culture medium, and incubation conditions. The positive controls mentioned are the blank control, negative mimic control, and negative inhibitor control.
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