Three kinds of siRNA (#1, #2 and #3) against human TMEM16A were designed and constructed by GenePharma (China). siRNA transfection of HUVECs was performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Negative siRNA (GenePharma) was used as negative control (siRNANC). The sequences of siRNA were as follows: siRNA #1, sense (5′- CCGGAGCACGAUUGUCUAUTT -3′), antisense (5′- AUAGACAAUCGUGCUCCGGTT -3′); siRNA #2, sense (5′- GGAAACAGAUGCGACUCAATT -3′), antisense (5′- UUGAGUCGCAUCUGUUUCCTT -3′); siRNA #3, sense (5′- GCUGUCAAGGAUCAUCCUATT -3′), antisense (5′- UAGGAUGAUCCUUGACAGCTT -3′); siRNANC, sense (5′- UUCUCCGAACGUGUCACGUTT -3′), antisense (5′- ACGUGACACGUUCGGAGAATT -3′). Briefly, cells at 70% confluent were starved with serum-free ECM for 1 h, and treated with siRNA/lipofectamin Opti MEM (Invitrogen) for 4–6 h. After cultured with complete ECM for 72 h, HUVECs were harvested for the following experiments.
Negative sirna
Manufactured by GenePharma
Sourced in China
Negative siRNA is a laboratory reagent used in gene silencing experiments. It serves as a control sample for comparison with experimental siRNA samples. Negative siRNA does not target any specific gene and is designed to have no known biological effects, providing a baseline for evaluating the effects of experimental siRNA treatments.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (3 mentions)
Sirnas, by GenePharma (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Si nc, by GenePharma (2 mentions)
Cell line nucleofector kit 5, by Lonza (1 mentions)
Sirna oligo, by GenePharma (1 mentions)
Si tlr4, by GenePharma (1 mentions)
Vinpocetine, by Merck Group (1 mentions)
Cse sirna, by GenePharma (1 mentions)
Indomethacin indo, by Merck Group (1 mentions)
Cse antibody, by Santa Cruz Biotechnology (1 mentions)
L ng nitroarginine methyl ester l name, by Merck Group (1 mentions)
Apamin, by Merck Group (1 mentions)
L cys, by Merck Group (1 mentions)
U87mg, by American Type Culture Collection (1 mentions)
U251mg, by American Type Culture Collection (1 mentions)
Heat inactivated fetal bovine serum, by Thermo Fisher Scientific (1 mentions)
Combimagnetofection, by Ozyme (1 mentions)
Ln308, by American Type Culture Collection (1 mentions)
18 protocols using negative sirna
1
HUVEC Transfection with TMEM16A siRNAs
2
Transfection of HUVECs with SelS Plasmid
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HUVECs were seeded into 6-well plates and prepared for transfection using Lipofectamine 3000 reagent (Invitrogen, USA). Briefly, 2 μg of empty vector plasmid or pcDNA3.1-SelS recombinant plasmid was mixed with 5 μl of Lipofectamine 3000 in serum-free medium. The mixture was dispensed into the wells for incubation. After a 6 h incubation, the cells were cultured in fresh medium for another 24 h and used for the subsequent assays. SelS siRNAs and negative siRNA (the base sequences are provided in Supplementary Table 1 ) were designed and synthesized by GenePharma Co. Ltd. (Shanghai, China). The steps for the transfection of the SelS siRNAs were the same as for the transfection of the pcDNA3.1-SelS plasmid, and the negative siRNA (Neg.RNA) served as a negative control.
3
Silencing KLK12 Gene in HT-29 Cells
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50 nmol/l small interfering RNA targeted KLK12 (siKLK12) and negative siRNA (Shanghai GenePharma Co., Ltd.) were inserted into pLKO.1-TRC vector (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences) to produce the recombinant plasmid. siRNA was used to transfect HT-29 cells using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The KLK12 coding region was inserted into the pLKO.1-TRC vector, and the HT-29 cells were transfected with KLK12 plasmid or empty vector. The cells that did not undergo transfection were used as a control. Sequences of the siRNAs used in the present study were as follows: KLK12 siRNA sense, 5′-AAACAG UGA CAG CCA CGU ATT-3′; KLK12 siRNA antisense, 5′-UAC GUG GCU GUC ACU GUU UGG-3′; negative siRNA sense, 5′-UUC UCC GAA CGU GUC ACG UTT-3′; negative siRNA antisense, 5′-ACG UGA CAC GUU CGG AGA ATT-3′. Transfected cells were cultured in complete medium for 48 h prior to use in the invasion, migration and apoptosis experiments.
4
Pokemon Overexpression Vector Construction
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We constructed a Pokemon overexpression vector according to the previous method [27 (link)], with minor modifications. Briefly, full-length human Pokemon was obtained by standard polymerase chain reaction (PCR) amplification from MCF7 cell cDNA and was cloned into the green fluorescence protein (GFP)-linked pcDNA3.1 plasmid. Pokemon small interfering RNA (siRNA) and negative siRNA were bought from GenePharma (Shanghai, China).
5
Knockdown of Autophagy Regulators in THP-1 Cells
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Both siRNA oligos against Beclin-1, RhoA, FIP200, and Rubicon genes and the negative siRNA were ordered from Shanghai GenePharma. siRNA sequences are listed in Supplementary Table 4 . siRNA oligos were transfected into THP-1 cells using Cell Line Nucleofector Kit V (Cat# VCA-1003, Lonza) following the manufacturer’s instructions.
6
siRNA-mediated TLR4 Knockdown
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Small interfering (si)RNA targeting toll-like receptor 4 (si-TLR4) and negative siRNA with a random sequence were synthesized by Shanghai GenePharma Co., Ltd., (Shanghai, China). The target sequences for TLR4 were: Sense, 5′-GGG CUU AGA ACA ACU AGA ATT-3′; and antisense, 5′-UUC UAG UUG UUC UAA GCC CTT-3′, and the sequences for the negative siRNA were: Sense, 5′-UUC UCC GAA CGU GUC ACG UTT-3′; and antisense, 5′-ACG UGA CAC GUU CGG AGA ATT-3′. The concentration used was 20 nM. All plasmids and oligonucleotides were transfected using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After 48 h, the cells were collected and subjected to subsequent experimentation.
7
Vascular Reactivity Modulation Assay
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CSE-siRNA and negative siRNA were purchased from GenePharma (Shanghai, China), and atelocollagen was purchased from KOKEN (Tokyo, Japan); CSE antibody was purchased from Santa Cruz (Delaware Ave, USA); NaHS, Acetylcholine(ACh), bradykinin, 9, 11-dideoxy-11α, 9α-epoxy-methanoprostaglandin F2α (U46619), and Vinpocetine were purchased from Sigma Chemicals (St. Louis, USA); lactate dehydrogenase (LDH) and malondialdehyde (MDA) assay kits were purchased from Nanjing Jiancheng Biological Co (Nanjing, China). ChTx, Apamin, L-Cys, L-NG-nitroarginine methyl ester (L-NAME), and indomethacin (Indo) were purchased from sigma Chemicals (St. Louis, USA); Krebs solution (comprising the following (mM): NaCl 118, KCl 3.4, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, and glucose 11.1) was aerated with a mixture of 95% O2 and 5% CO2 and oxygenated during the incubation period.
8
Silencing DcR3 in Malignant Glioma Cells
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Three malignant glioma cell lines (U251MG, LN-308, and U87MG) of humans were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). These three cell lines were incubated in Dulbecco’s Modified Eagle Medium (DMEM). All cell lines were supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen Corp Grand Island, NY, USA) under a humidified 5% CO2 atmosphere with 2 mM glutamine and gentamicin at 37°C. Four DcR3-specific siRNAs (Table 1 ) were synthesized by GenePharma (ShangHai, People’s Republic of China) and merged into one siRNA pool. Five groups were designed in the current study: blank control, mock control, negative siRNA control, scramble siRNA, and DcR3-specific siRNA. Blank control groups were cultured with only serum-free DMEM medium without any transfection reagents or siRNA. In the mock control groups, only transfection reagent was added. Negative control groups were treated with negative siRNA (GenePharma). The sequences of DcR3 siRNAs were scrambled and rearranged as another control (Scramble siRNAs). CombiMAGnetofection (OZ Biosciences, Marseille cedex 9, France) was used for the transfection. All the in vitro experiments were performed in triplicate.
9
Role of NOX4 in Hyperglycemia-Induced Cellular Responses
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Experiments were performed with selected specific small interfering RNA sequence as follows (siRNAs; GenePharma, Shanghai, China), Nox4: sense 5′-CCAGUGGUUUGCAGAUUUATT-3′, antisense 5′-UAAAUCUGCAAACCACUGGTT-3′; negative-siRNA: sense 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense 5′-ACGUGACACGUUCGGAGAATT-3; FAM-siRNA: sense 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense 5′-ACGUGACACGUUCGGAGAATT-3′ as positive control. siRNA conditions were optimized. NOX 4 siRNA was transfected by the Lipofectamine 2000 transfection reagent (Invitrogen, U.S.A.) according to the manufacturer’s protocol. Cells were used for experiments at 48 h after transfection.
Cells were randomly grouped into the following four groups: (1) without NOX 4 siRNA and HG (Glu-, NOX4 siRNA-); (2) with NOX 4 siRNA but without HG (Glu-, NOX4 siRNA+); (3) with HG, but without NOX4 siRNA (Glu+, NOX4 siRNA-); (4) with HG and NOX4 siRNA (Glu+, NOX4 siRNA+).
Cells were randomly grouped into the following four groups: (1) without NOX 4 siRNA and HG (Glu-, NOX4 siRNA-); (2) with NOX 4 siRNA but without HG (Glu-, NOX4 siRNA+); (3) with HG, but without NOX4 siRNA (Glu+, NOX4 siRNA-); (4) with HG and NOX4 siRNA (Glu+, NOX4 siRNA+).
10
PHGDH Knockdown Impacts Cell Colony Formation
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PHGDH knockdown was performed by transfecting cells with independent siRNA candidates, siPHGDH#2, siPHGDH#4 and siPHGDH#5 (GenePharma, China) using Lipofectamine 3000 reagent (Invitrogen, CA) following the protocol, negative siRNA (GenePharma) as mock control. The sense sequences of siPHGDHs were as following: siPHGDH#2 (UGC CGG AAG AUC UUG CAA GTT), siPHGDH#4 (GGG AAC AGA GCU GAA UGG ATT), siPHGDH#5 (CUG ACC CUG UAG UAC AGC ATT). PHGDH expression were then detected by immunoblotting after 72 h transfection.
The transfected cells were replated on the 6-well plates at 500 cells/well, and cultured in complete medium for about 2 weeks. Cells were then stained with crystal violet and individual colonies (>50 cells) were counted.
The transfected cells were replated on the 6-well plates at 500 cells/well, and cultured in complete medium for about 2 weeks. Cells were then stained with crystal violet and individual colonies (>50 cells) were counted.
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