The miR-7 mimic/inhibitor, pcDNA3.1-cdc42, si-cdc42, and their respective control were chemically synthesized or purchased from GenePharma Co. (Shanghai, China) and transfected (100 nM) or co-transfected into NSCs cells using Invitrogen Lipofectamine™ 2000 transfection reagent (Thermo Fisher Scientific). The detailed information regarding miR-7 mimic and miR-7 inhibitor and their controls is listed as follows: miR-7 mimic: (sense: 5′ -UGGAAGACUAGUGAUUUUGUUGU-3′, antisense: 5′ -AACAAAAUCACUAGUCUUCCAUU-3′); the negative control of miR-7 mimic (pre-NC, sense: 5′ -UUCUCCGAACGUGUCACGUTT-3′, antisense: 5′ -ACGUGACACGUUCGGAGAATT-3′); miR-7 inhibitor: (5′ -ACAACAAAAUCACUAGUCUUCCA- 3′), the negative control of miR-7 inhibitor (NC, 5′ -CAGUACUUUUGUG UAGUACA A-3′).
Invitrogen lipofectamine 2000 transfection reagent
Manufactured by Thermo Fisher Scientific
Sourced in China
Invitrogen Lipofectamine™ 2000 is a transfection reagent designed to facilitate the delivery of nucleic acids, such as plasmid DNA, siRNA, and mRNA, into a variety of mammalian cell types. It is a lipid-based reagent that forms complexes with the nucleic acids, which can then be taken up by the target cells.
Automatically generated - may contain errors
4 protocols using invitrogen lipofectamine 2000 transfection reagent
1
miR-7 Modulation in Neural Stem Cells
2
Functional Analysis of linc-UBC1 in CRC Cells
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The functional relevance of linc-UBC1 in CRC cell lines was investigated using linc-UBC1-specific small interfering RNAs (siRNAs). The nucleotide sequences of the siRNAs were as follows: si-linc-UBC1-1, 5′-GGUUUCUGCCCUCAUCCUU-3′; si-linc-UBC1-2, 5′-GCUUCUAGUCCUCUCCUUA-3′; si-linc-UBC1-3, 5′-GCUGGAACCCAUUUACUAA-3′; and negative control (si-NC), 5′-CGUGGGUGGAUGCAUGGAUTT-3′. siRNAs were chemically synthesized by GenePharma Co. (Shanghai, China) and transfected into SW620 cells using Invitrogen Lipofectamine™ 2000 transfection reagent (catalog no 11668-019; Thermo Fisher Scientific) according to the manufacturer’s protocol. The silencing efficiency was evaluated at 48 h after transfection using RT-qPCR. The siRNA that demonstrated the most significant silencing capacity was further utilized for functional investigations in the cell line.
3
siRNA-mediated Silencing of Na+,K+-ATPase
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HK-2 cells were seeded on 6-well cell culture plates at density of 1 × 105 cells/well a day prior transfection to achieve 80% confluency at the time of transfection. For the last 6 h the cells were grown in medium without antibiotics. The cells were transfected with siRNA against human Na+,K+-ATPase α1 mRNA (ATP1A1, No. L-006111-00-0005) or human Na+,K+-ATPase α3 mRNA (ATP1A3, No. J-004614-00-0005) (both ON-TARGETplus–SMARTpool from Horizon Discovery) using Invitrogen Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s guidelines with siRNA in the final concentration of 5 nM. To test the off-target effects of siRNA, a non-targeting siRNA pool (ON-TARGET plus Non-targeting Pool, No. D-001810-10-20, Horizon Discovery) was used. After 24 h of transfection the medium was replaced with fresh DMEM (10% FBS, without antibiotics/siRNA) and the cells were incubated for another 24 h. To enable measurement of lactate secretion into medium, the cells were grown in serum-free DMEM for the last 8 h. The cells were washed with PBS and frozen at − 80 °C until further analysis. Gene knock-down was assayed using qPCR and immunoblot.
4
Glioma Cell Line Transfection and Irradiation
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Human glioma U251, U-118MG and SHG-44 cell lines were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and cultured in DMEM supplemented with 10% dialyzed fetal bovine serum (FBS) and 1% PS (100 U/ml penicillin and 100 µg/ml streptomycin). Cells were maintained in a humidified incubator with 5% CO2 at 37°C. The cells were transfected with pcDNA3/hsa-miR-212 or the pcDNA3 control, antisense oligonucleotide (ASO)-miR-212 or ASO-ctrl, pcDNA3/EGFP, pcDNA3/EGFP-BRCA1, or pcDNA3/EGFP-BRCA1 3′-UTR mut using Invitrogen Lipofectamine™ 2000 transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. Cells were exposed to different doses of 137Cesium γ-irradiation at a rate of 0.8 Gy/min.
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