ARNO siRNA (Mm_Pscd2_3) or a negative non-specific siRNA control (Allstars siRNA) were transfected into SFs using HiPerFect transfection reagent (all Qiagen, UK) according to the manufacturer’s protocol. Briefly, siRNAs were diluted in 8% HiPerFect transfection reagent in DMEM (Invitrogen, UK) and incubated for 10-15 minutes at room temperature to form transfection complexes before adding to the cells (final siRNA concentration 10 nM). Cells were incubated with transfection complex for 24 hours and then washed with complete DMEM. Cells were transfected again using this protocol after 3 days, prior to experimental analysis as indicated.
Hiperfect transfection reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
The HiPerFect Transfection Reagent is a lipid-based transfection reagent designed to efficiently deliver nucleic acids into a variety of cell types. It facilitates the uptake and expression of plasmid DNA, siRNA, and other nucleic acids in both adherent and suspension cell cultures.
Automatically generated - may contain errors
Lab products found in correlation
Hiperfect transfection reagent, by Qiagen (9 mentions)
Opti mem 1 reduced serum medium, by Thermo Fisher Scientific (3 mentions)
Dmem glutamax, by Thermo Fisher Scientific (2 mentions)
Antibiotic antimycotic solution, by Merck Group (2 mentions)
Opti mem reduced serum medium, by Thermo Fisher Scientific (2 mentions)
On targetplus smartpool sirna, by Horizon Discovery (1 mentions)
Sirnas, by Qiagen (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Inhibitor nc, by GenePharma (1 mentions)
Mimic nc, by GenePharma (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Insect xpress medium, by Lonza (1 mentions)
Panc 1, by American Type Culture Collection (1 mentions)
Bxpc 3, by American Type Culture Collection (1 mentions)
Aspc 1, by American Type Culture Collection (1 mentions)
Mia paca 2, by American Type Culture Collection (1 mentions)
Su 86, by American Type Culture Collection (1 mentions)
Hiperfect reagent, by Qiagen (1 mentions)
Dulbecco s modified eagle s medium f12, by Thermo Fisher Scientific (1 mentions)
15 protocols using hiperfect transfection reagent
1
siRNA Transfection in Synovial Fibroblasts
2
PRRSV Infection of MARC-145 and PAM Cells
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MARC-145 cells [5 (link)] and HEK293T were obtained from ATCC and grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The PAM cells were harvested from PRRSV or PBS inoculated three-week-old PRRSV-negative pigs (n = 5 per group) in a previous study [10 (link)], which was approved by the Laboratory Animal Administration Committee of Xi’an Jiaotong University (Approval #: XJTULAC2016-318) according to the Guidelines for Animal Experimentation of Xi’an Jiaotong University and Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication NO. 85-23, revised 2011). PRRSV HuN4 strain [24 (link)], a highly pathogenic PRRSV (HP-PRRSV) strain, was used to inoculate MARC-145 cells at a multiplicity of infection (MOI) of 1. Virus titers were determined in MARC-145 cells for the median tissue culture infectious dose (TCID50), as described previously [25 (link)].
Lipofectamine™ 2000 Transfection Reagent (Cat. No. 11668-027, Invitrogen, Waltham, MA, USA) and HiPerFect Transfection Reagent (Cat. No. 301704, QIAGEN, Hilden, Germany) were used to transfect plasmid DNA and siRNA, respectively, into the cells according to the manufacturer’s instructions.
Lipofectamine™ 2000 Transfection Reagent (Cat. No. 11668-027, Invitrogen, Waltham, MA, USA) and HiPerFect Transfection Reagent (Cat. No. 301704, QIAGEN, Hilden, Germany) were used to transfect plasmid DNA and siRNA, respectively, into the cells according to the manufacturer’s instructions.
3
Chk2 Knockdown in A9 Cells
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ON-TARGET plus SMART pool siRNAs directed against mouse Chk2 (cat # L-0406034-00) was obtained from Dharmacon. A9 cells plated in isoleucine-deprived media in 60 mm dishes were transfected at the day of plating with 40 nm of siRNA using HiPerfect transfection reagent (Invitrogen). Transfections were repeated 24 hours later and the next day the cells were released into complete media and processed as described in the figure legends.
4
siRNA-Mediated Knockdown of TFEB
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Chemically synthesized, double-stranded small interfering RNAs (siRNAs) for TFEB and control were purchased from Qiagen. The siRNA sequences targeting TFEB and control are 5′-CTCGACGTTCTGCAAGGTCTA-3′ and 5′-AATTCTCCGAACGTGTCACGT-3′, respectively. Forty to 50% confluent A549 cells (1 × 106 cells/well) in the wells on a six-well plate, split at least 24 h before transfection, were transfected using the HiPerfect transfection reagent (Qiagen, Spain) according to a modified version of the manufacturer’s instructions. Briefly, 6 µl of HiPerfect transfection reagent was added to 100-µl mixtures of siRNAs (10 nM) and Optimem (Invitrogen, Spain), mixed by inversion, and incubated for 15 min at room temperature. The entire transfection mixture was added to A549 cells containing fresh serum-free Optimem. After A549 cells were incubated for 4 h at 37°C, an additional 3 ml of DMEM supplemented with FBS and antibiotics was added to each well on the plate. After additional incubation for 48 h, TFEB expression was studied by Western blotting.
5
Transfection of miR-325-3p mimic and inhibitor
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miR-325-3p mimic (5′-AACTATCCTCCAGGAGTTATTT-3′) or inhibitor (5′-AAATAACTCCTGGAGGATAGTT-3′) and the respective negative controls (NCs; NC mimic, 5′-TTCTCCGAACGTGTCACGT-3′; NC inhibitor, 5′-TTCTCCGAACGTTGTCACGT-3′) (all from Shanghai GenePharma Co., Ltd.) are chemically modified analogs, which can be transfected into cells without using a vector. Transfection was performed using HiPerFect Transfection Reagent (Qiagen GmbH), as previously described (16 (link)). Briefly, HT-29 cells were seeded in a 6-well plate at a density of 106 cells/well. Subsequently, the cells were transfected with miR-325-3p mimic, inhibitor or NC for 48 h using HiPerFect Transfection Reagent according to the manufacturer's instructions. A total of 12 µl HiPerFect Transfection Reagent was mixed with 100 µl cell culture in serum-free DMEM (Invitrogen; Thermo Fisher Scientific, Inc.). Meanwhile, 10 µl miR-325-3p mimic, inhibitor or NC was mixed with serum-free DMEM. Then, the two mixtures were mixed and incubated at room temperature for 15 min. After that, the mixture was added in the 6-well plate at a final concentration of 20 nM. Following transfection for 48 h, the cells were collected for subsequent experiments.
6
VSMC siRNA Transfection Protocol
7
CDK7 Silencing in MCF-7 and LCC2 Cells
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The MCF-7 and LCC2 cells were plated in six-well plates and seeded at density 2 × 105/well. Twenty-four hours after seeding, cells reached 50%–60% confluence and were transfected with 25–200 nM siRNA targeting CDK7 for 72 h. Control plates included no treatment, and treatment with scramble non-targeting siRNA was transfected. Two different siRNAs (siRNA-CDK7-1 and siRNA-CDK7-2; Sigma–Aldrich) with the following using HiPerfect transfection reagent (Invitrogen) following protocols provided by the manufacturer. After 72 h, cells were collected for protein extraction or RNA isolation for RT-PCR. First, the two siRNA were studied to confirm its silencing effect on CDK7 using Western blot and RT-PCR with different concentrations.
8
Interferon Stimulation and Protein Turnover
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HEK293T cells (ATCC) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
For interferon stimulation, recombinant human IFNα-2a (Cat. No. Z03003-50, GenScript) was added to the cultured cells at a final concentration of 500 U/mL (unless stated differently in section “Results” and the figure legends). The cells were harvested at time points indicated in the results or the figure legends for further analysis.
MG132 (Cat. No. S1748, Beyotime Biotechnology), a proteasome inhibitor, was added to cells at 10 μM final concentration for 6 h before the cells being harvested for further analysis. Cycloheximide (CHX) (Cat. No. SC0353, Beyotime Biotechnology) was added to cultured cells at a final concentration of 100 μg/mL to block protein translation to determine the half-life of JAK2. The cells were harvested at the time points indicated in section “Results” or the figure legends for immunoblotting.
LipofectamineTM 2000 Transfection Reagent (Cat. No. 11668-027, Invitrogen) and HiPerFect Transfection Reagent (Cat. No. 301704, QIAGEN) were used to transfect plasmid DNA and siRNA, respectively, into the cells according to the manufacturers’ instructions.
For interferon stimulation, recombinant human IFNα-2a (Cat. No. Z03003-50, GenScript) was added to the cultured cells at a final concentration of 500 U/mL (unless stated differently in section “Results” and the figure legends). The cells were harvested at time points indicated in the results or the figure legends for further analysis.
MG132 (Cat. No. S1748, Beyotime Biotechnology), a proteasome inhibitor, was added to cells at 10 μM final concentration for 6 h before the cells being harvested for further analysis. Cycloheximide (CHX) (Cat. No. SC0353, Beyotime Biotechnology) was added to cultured cells at a final concentration of 100 μg/mL to block protein translation to determine the half-life of JAK2. The cells were harvested at the time points indicated in section “Results” or the figure legends for immunoblotting.
LipofectamineTM 2000 Transfection Reagent (Cat. No. 11668-027, Invitrogen) and HiPerFect Transfection Reagent (Cat. No. 301704, QIAGEN) were used to transfect plasmid DNA and siRNA, respectively, into the cells according to the manufacturers’ instructions.
9
TDP-43 Knockdown in SK-N-BE Neuroblastoma
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SK-N-BE neuroblastoma cell line was cultured in standard conditions in DMEM-Glutamax (#31966-021, Thermo Fisher Scientific) supplemented 10% fetal bovine serum and 1 × antibiotic-antimycotic solution (#A5955; Sigma). RNA interference of TDP-43 was achieved using HiPerfect Transfection Reagent (#301705, Qiagen) and siRNA specific for human TDP43 (5′-gcaaagccaagaugagccu-3′); as control siRNA for Luciferase was used (5′-uaaggcuaugaagagauac-3′; Sigma). Immediately before transfection 2–4 × 105 cells were seeded in 6-well plates in 1.4 ml of medium containing 10% fetal serum. A volume of 3 µl of each siRNA (40 µM solution in water), was added to 91 µl of Opti-MEM I reduced serum medium (#51985-026, Thermo Fisher Scientific), incubated 5 minutes at room temperature and subsequently 6 µl of HiPerfect Transfection Reagent were added. The silencing procedure was performed again after 24 and 48 hours.
10
Silencing TDP-43 in SH-SY-5Y Cells
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SH-SY-5Y neuroblastoma cell line was cultured in standard conditions in DMEM-Glutamax (#31966-021, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum and 1 × antibiotic-antimycotic solution (#A5955; Sigma). RNA interference of TDP-43 was achieved using HiPerfect Transfection Reagent (#301705, Qiagen) and siRNA specific for human TDP43 (5′-gcaaagccaagaugagccu-3′); as control, siRNA for luciferase was used (5′-uaaggcuaugaagagauac-3′; Sigma). Immediately before transfection, 2–4 × 105 cells were seeded in 6-well plates in 1.4 ml of medium containing 10% fetal serum. A volume of 3 μl of each siRNA (40 μM solution in water) was added to 91 μl of Opti-MEM I reduced serum medium (#51985-026, Thermo Fisher Scientific) and incubated 5 min at room temperature, and subsequently, 6 μl of HiPerfect Transfection Reagent was added. The silencing procedure was performed again after 24 and 48 h.
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