To investigate the effects of using small interfering (si)RNA to downregulate ZFAS1 expression, TT and SW579 cells were divided into the following groups: i) Control (without transfection); ii) si-Control (transfected with 50 nM si-Control); and iii) si-ZFAS1 (transfected with 50 nM si-ZFAS1). The si-Control (5′-UUCUCCGAACGUGUCACGUTT-3′) and si-ZFAS1 (5′-CUAACUGCCUACCUGCAUATT-3′) were obtained from Shanghai GenePharma Co., Ltd., and the cells were transfected using Lipofectamine® 3000 (cat. no. L3000015; Thermo Fisher Scientific, Inc.). To specify the linkage between miR-302a-3p and ZFAS1 on the hallmarks of thyroid carcinoma, TT and SW579 cells were divided into the following groups: i) Control (without transfection); ii) inhibitor-negative control (NC; transfected with 50 nM miR-302a-3p inhibitor-NC; iii) inhibitor (transfected with 50 nM miR-302a-3p inhibitor); iv) inhibitor + si-ZFAS1 (transfected with 50 nM miR-302a-3p inhibitor and 50 nM si-ZFAS1); and v) si-ZFAS1 (transfected with 50 nM si-ZFAS1). The miR-302a-3p inhibitor-NC (5′-CAGUACUUUUGUGUAGUACAA-3′) and miR-302a-3p inhibitor (5′-UCACCAAAACAUGGAAGCACUUA-3′) were obtained from Shanghai GenePharma Co., Ltd, and the cells (2×104 cells/well; 96-well plate) were transfected using Lipofectamine® 3000 (cat. no. L3000015; Thermo Fisher Scientific, Inc.). The cells were cultured for 24 h prior to subsequent experimentation.
Lipofectamine 3000
Manufactured by GenePharma
Sourced in China
Lipofectamine 3000 is a cationic lipid-based transfection reagent designed for efficient delivery of DNA, RNA, or other nucleic acids into a variety of mammalian cell types. It facilitates the uptake of genetic material into cells, enabling researchers to study gene expression, knockdown, or other applications.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (15 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Si nc, by GenePharma (3 mentions)
Dual luciferase reporter assay system, by Promega (3 mentions)
Mimic nc, by GenePharma (3 mentions)
Inhibitor nc, by GenePharma (2 mentions)
Aml12 cells, by American Type Culture Collection (2 mentions)
Hepg2, by American Type Culture Collection (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Si control, by GenePharma (1 mentions)
Sizfas1, by GenePharma (1 mentions)
Opti mem culture medium, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Rle 6tn cells, by American Type Culture Collection (1 mentions)
Oxycycler c42 system, by Biospherix (1 mentions)
Prl tk, by Promega (1 mentions)
Chemiluminescence meter, by Promega (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Nc duplex, by GenePharma (1 mentions)
65 protocols using lipofectamine 3000
1
Investigating ZFAS1 Downregulation in Thyroid Carcinoma
2
PTX3 Overexpression in Cell Induction
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The blank group was cultured with normal medium. The induced group was cultured with induction reagent I. The short interfering RNA (siRNA) control group was cultured with induction reagent I in Opti-MEM culture medium (1734724; Gibco, USA) containing Lipofectamine®3000 (1769228; Invitrogen, Shanghai, China) and NC (GenePharma, Shanghai, China). The siRNA group was cultured with induction reagent I in Opti-MEM culture medium containing Lipofectamine®3000 and siRNA (GenePharma, Shanghai, China). The PTX3 overexpression plasmid control group was cultured with induction reagent I in Opti-MEM culture medium containing Lipofectamine® 3000, PM3000 diluent, and the PTX3 empty vector (GenePharma, Shanghai, China). The PTX3 overexpression plasmid group was cultured with induction reagent I in Opti-MEM culture medium containing Lipofectamine®3000, P3000™ diluent (1769230; Invitrogen; Shanghai, China), and the PTX3 overexpression plasmid (GenePharma, Shanghai, China: NM_008987.3).
3
Transfection of Cervical Cancer Cells
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The cervical cancer cells were transfected with control cDNA or CBX7 cDNA or control siRNA or CBX7 siRNAs (Genepharma, Shanghai, China) via Lipofectamine 3000, following the instruction’s protocol.33 (link) The sequence of CBX7 siRNA was as follows: 5′-CAC CTT GCA TGC ACC TTG CTA-3′. Then, the transfected cells were seeded into 96-well or six-well plates to analyze further, as described in Results .
4
Intermittent Hypoxia Regulation of RLE-6TN Cells
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RLE-6TN cells (ATCC, Manassas, Virginia, USA) were cultured in DMEM/F12 medium consisting of 10% FBS, 100 μg/mL streptomycin, and 100 U/mL penicillin (Sigma–Aldrich, Merck KGaA, Darmstadt, Germany). The treated cells were cultured at 37 °C with 5% CO2 in air.
The intermittent hypoxia (IH) regimen consisted of alternative cycle of 20 min hypoxia (1% O2 and 5% CO2) and 20 min reoxygenation (21% O2 and 5% CO2), with 40 min for each cycle and a total 72 cycles and was conducted on the OxyCycler C42 system (BioSpherix, Redfield, NY, UAS). Then, RLE-6TN cells were treated with 20 μg ADSCs-EVs for 24 h before IH regimen, with GW treatment as the control [22 (link)]. In addition, RLE-6TN cells were cultured in the 6-well plates at a density of 1 × 106 cells/per well. RLE-6 TN cells were transfected with KDM6B pcDNA3.1 (oe-KDM6B) or HMGA2 pcDNA3.1 (oe-HMGA2) or the negative control (GenePharma, Shanghai, China) by means of Lipofectamine 3000. After 24 h, the transfection efficiency was tested or RLE-6TN cells were exposure to a specified number of IH cycles.
The intermittent hypoxia (IH) regimen consisted of alternative cycle of 20 min hypoxia (1% O2 and 5% CO2) and 20 min reoxygenation (21% O2 and 5% CO2), with 40 min for each cycle and a total 72 cycles and was conducted on the OxyCycler C42 system (BioSpherix, Redfield, NY, UAS). Then, RLE-6TN cells were treated with 20 μg ADSCs-EVs for 24 h before IH regimen, with GW treatment as the control [22 (link)]. In addition, RLE-6TN cells were cultured in the 6-well plates at a density of 1 × 106 cells/per well. RLE-6 TN cells were transfected with KDM6B pcDNA3.1 (oe-KDM6B) or HMGA2 pcDNA3.1 (oe-HMGA2) or the negative control (GenePharma, Shanghai, China) by means of Lipofectamine 3000. After 24 h, the transfection efficiency was tested or RLE-6TN cells were exposure to a specified number of IH cycles.
5
Dual-Luciferase Assay for miRNA-27a and GSK3β 3'UTR Interaction
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GV148-GSK3β-3′UTR, GV148-GSK3β-3’UTR-mut, GV272-miR-27a promoter-wild and GV272-miR-27a promoter-mut plasmids were obtained from GENECHEM (Shanghai, China). HEK293T or Hep2 cells seeded in 96-well plate in triplicate were cotransfected with GV148-GSK3β-3′UTR, GV148-GSK3β-3′UTR-mut, GV272-miR-27a promoter-wild or GV272-miR-27a promoter-mut plasmids and miRNA-27a mimic, si-RARα or non-relative control RNA duplex (NC duplex; GenePharma) by using Lipofectamine™ 3000 in accordance with the manufacturer's procedure, respectively. pRL-TK (Promega, Madison, WI, USA) was transfected as a normalization control. Cells were collected at 24 h after transfection and luciferase activity was measured using a dual-luciferase reporter assay kit (Promega) and recorded by Chemiluminescence Meter (Promega).
6
miR-126 Mimic Transfection and OGD/R Assay
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miR-126 mimic (mimic-miR; 5′-CATTATTACTTTTGGTACGCG-3′) and mimic-NC (cat. no. B04001) were purchased from Shanghai GenePharma Co., Ltd. Lipofectamine® 3000 (cat. no. L3000075; Invitrogen; Thermo Fisher Scientific, Inc.) was used to transfect 0.5 pM mimic-miR and 0.5 pM mimic-NC into 1×106 SH-SY5Y cells according to the manufacturer's instructions. The transfected cells (24 h after transfection) were then subjected to OGD/R treatment. Untransfected SH-SY5Y cells that underwent OGD/R treatment alone (Model group) were also used as a control. Each assay was only performed once, but with three wells.
7
Regulation of PTEN by miR-494 in AML12 cells
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Murine hepatic cell line AML12 cells (ATCC, Manassas, VA, U.S.A.) were cultured in DMEM/Ham’s F12 medium supplemented with 10% FBS, 100 u/ml penicillin, 100 μg/ml streptomycin, 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium, and 40 ng/ml dexamethasone. AML12 cells at 70% confluence in six-well plates were transfected with 50 nM of miR-494 mimics, miR-494 inhibitor, mimics NC, or inhibitor NC (GenePharma Biotech) using Lipofectamine 3000 transfection agent. After 48 h, total RNA and protein were extracted, and PTEN mRNA and protein expression was analyzed by quantitative reverse-transcription PCR (qRT-PCR) and Western blot as described above.
8
Plasmid Transfection and siRNA Knockdown
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The PKM2 and SIRT3 expression plasmids, as well as the PKM2 mutant plasmid, were synthesized by GenePharma (Shanghai, China). The SIRT3-H248Y plasmid was obtained from Addgene (Watertown, MA, USA). Briefly, A549 cells were transfected with 2 μg of the expression plasmid or the negative control vector using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). For siRNA transfection, cells were transfected with specific or control siRNAs (GenePharma) using Lipofectamine 3000 for 48 h. The sequences of the siRNAs were as follows: SIRT3, sense 5’-GAA ACU ACA AGC CCA ACG UTT-3’ and antisense 5’-ACG UUG GGC UUG UAG UUU CTT-3’; PKM2 sense 5’- CAU CUA CCA CUU GCA AUU ATT-3’ and antisense 5’- UAA UUG CAA GUG GUA GAU GTT-3’; and negative control, sense 5’-UUC UCC GAA CGU GUC ACG UTT-3’ and antisense 5’ ACG UGA CAC GUU CGG AGA ATT-3’.
9
Silencing TTN-AS1 and miR-211-5p in TNBC
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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).
10
MiR-21-5p and TSP-1 Regulation in EPCs
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To regulate the expressions of miR-21-5p and TSP-1 in EPCs, miR-21-5p mimic, mimic-negative control (NC), TSP-1 siRNAs, and siRNA control were transfected into cells with Lipofectamine 3000, in accordance with the manufacturer’s instructions (GenePharma, Shanghai, China). The primer sequences are shown in Table 2 . After 72 h of incubation, the EPCs were harvested for subsequent experiments.
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