Two human glioblastoma cell lines U251 and T98G (Cell bank, Chinese Academy of Science) were used. Cells were cultured cultivated in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin in an incubator at 37 °C with CO2 and humidity set to 5% and 95%, respectively. HLA-F-AS1 and MEG3 were overexpressed in cells by transfecting pcDNA-HLA-F-AS1 or -MEG3 expression vector through transient transfections mediated by Neon Electroporation Transfection (Thermo Fisher Scientific). All operations were performed following the manufacturer’s instructions.
Neon electroporation transfection
Manufactured by Thermo Fisher Scientific
The Neon Electroporation Transfection is a lab equipment product designed for efficient delivery of nucleic acids, such as DNA or RNA, into a variety of cell types. It utilizes electroporation technology to create temporary pores in the cell membrane, allowing the introduction of genetic material.
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4 protocols using neon electroporation transfection
1
Overexpression of HLA-F-AS1 and MEG3 in Glioblastoma Cells
2
Overexpression of circCTNNA1 and miR-34a in MCL Cells
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Two human MCL cell lines JVM-2 and Z138 were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). Cells were cultivated in DMEM (GIBCO) containing FBS (10%), penicillin (50 units/mL) and streptomycin (50 mg/mL) in an incubator with temperature, humidity and CO2 set to 37 °C, 95% and 5%, respectively.
Overexpression of circCTNNA1 and miR-34a was achieved in cells using Neon Electroporation Transfection (Thermo Fisher Scientific) to transfect circCTNNA1-pcDNA3.1 vector or miR-34a mimic. The dosages of vector and miRNA were 50 and 150 μM for 107 cells, respectively. The amount of vector and miRNA was blow 10% of the total volume.
Overexpression of circCTNNA1 and miR-34a was achieved in cells using Neon Electroporation Transfection (Thermo Fisher Scientific) to transfect circCTNNA1-pcDNA3.1 vector or miR-34a mimic. The dosages of vector and miRNA were 50 and 150 μM for 107 cells, respectively. The amount of vector and miRNA was blow 10% of the total volume.
3
Overexpression of BRCAT54 and miR-1269b in Endothelial Cells
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Hemangioma-derived endothelial cells (HDECs) and human umbilical vein endothelial cells (HUVECs) were bought from the Cell Bank of Chinese Academy Sciences (Shanghai, China) and were cultivated in DMEM (Gibco) containing fetal bovine serum (10%) and penicillin–streptomycin (1%). Cell culture was carried out in an incubator with 5% CO2 and with temperature and humidity set to 37 ºC and 95%, respectively.
To overexpress BRCAT54 and miR-1269b, cells were transfected with miR-1269b mimic and/or pcDNA 3.1- BRCAT54 vector through transient transfections achieved with Neon Electroporation Transfection (Thermo Fisher Scientific). In each transfection, 106 cells were transfected with 30 mM vector and/or 80 mM miRNA. The confirmation of overexpression (compared to NC mimic or empty vector transfection group) was performed every 24 h until the end of in vitro cell experiments.
To overexpress BRCAT54 and miR-1269b, cells were transfected with miR-1269b mimic and/or pcDNA 3.1- BRCAT54 vector through transient transfections achieved with Neon Electroporation Transfection (Thermo Fisher Scientific). In each transfection, 106 cells were transfected with 30 mM vector and/or 80 mM miRNA. The confirmation of overexpression (compared to NC mimic or empty vector transfection group) was performed every 24 h until the end of in vitro cell experiments.
4
Overexpression of circ-MYBL2 and miR-28 in Lung Cancer Cells
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H1299 and A549 cells were transfected with either pcDNA3.1- circ-MYBL2 vector or miR-28 mimic (Invitrogen, China). Briefly, a 6-well plate was seeded with these cells at a density of 106 cells per well, and cell culture was performed overnight. After that, Neon Electroporation Transfection (Thermo Fisher Scientific) was used to transiently transfect vector or miRNA into cells. Cells were washed with fresh medium at 6 h after transfection, followed by cell culture in fresh medium for another 48 h. The same system was also applied to transfect negative control (NC) miRNA and empty vector as the control.
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