Gene silencing of HIF-1α and miR-210 was induced overnight (12 h) by adenoviral transfection (100 nM of the appropriate siRNA with 2.5 μg/ml TransFast transfection reagent (Promega, Madison, WI, USA)) HIF-1α siRNA in encoded sequence 5'-CCA CCA CUG AUG AAU UAA AUU TT-3 '(Microsynth, Oligo ID # 2,808,643), and siRNA miR-210 in the coding sequence 5'-UCA GCC GCU GUC ACA CGC ACA GTT-3' (Microsynth, Oligo ID # 2,808,645), and negative siRNA control (Thermofisher, # 4,390,843) on A375 malignant melanoma subconfluent cells. The genetically encoded biosensors Peredox-mCherry, and MitoTracker Red CMXRos were transfected with the appropriate plasmid DNA Peredox-mCherry (pcDNA3.1), and MitoTracker Red CMXRos (tetramethylrhodamine methyl ester and 10-N-nonyl acridine orange) into A375 subconfluent cells. Cells which were treated with negative siRNA control (siR neg cont) were used as control. The cells were used for analysis after 48 h of transfection.
Negative sirna control
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Negative siRNA Control is a non-targeting control small interfering RNA (siRNA) designed to have no known mRNA targets in the cell. It is used as a control to determine the specific effects of target gene silencing using RNAi technology.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (3 mentions)
Siaurka, by Thermo Fisher Scientific (2 mentions)
Sieif4e, by Thermo Fisher Scientific (2 mentions)
Lipojet reagent, by SignaGen (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Scrambled control, by Thermo Fisher Scientific (2 mentions)
Anti hsa mir 24, by Thermo Fisher Scientific (2 mentions)
Pre hsa mir 24, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Transfast transfection reagent, by Promega (1 mentions)
17 dmag, by InvivoGen (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Sirnas against, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit with rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (1 mentions)
Aβ1 42, by AnaSpec (1 mentions)
Anhydrous dimethyl sulfoxide dmso, by Merck Group (1 mentions)
Aβ1 42 hilyte 488, by AnaSpec (1 mentions)
Taqman openarray mouse phagocytosis panel, by Thermo Fisher Scientific (1 mentions)
8 protocols using negative sirna control
1
Silencing HIF-1α and miR-210 in A375 Melanoma
2
Macrophage Activation and Cytokine Profiling
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Mouse bone marrow–derived macrophages (BMDMs) were generated as described previously.(22 ) BMDMs (0.8 million cells/well) were stimulated with 100 ng/mL lipopolysaccharide (LPS; Sigma‐Aldrich, St. Louis, MO), 25 mM ethanol, 1 μM 17‐dimethylaminoethylamino‐17‐demethoxygeldanamycin (17‐DMAG; Invivogen, San Diego, CA), and 0.5 μM PU‐WS13,(23 ) as indicated. RAW 264.7 cells were transiently transfected with GP96 siRNA (50 nM or 100 nM) or negative siRNA control (Thermo Fisher Scientific, Waltham, MA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) for 48 hours and then stimulated with LPS for the final 6 hours. RNA, culture media, and whole cell lysates were collected for the indicated assays. BMDMs were also collected from LPS‐injected WT and M‐GP96KO mice for cytokine expression analysis using RT‐PCR.
3
Targeting HIF1α and PFKFB3 in hiPSC-CMs
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siRNAs against HIF1α (siRNA ID: s6539, 4390824) and PFKFB3 (siRNA ID: s10359, 4390824), respectively, and a negative siRNA control (4390843) were purchased from Thermo Fisher (Waltham, MA). hiPSC-CMs were pre-treated with the above siRNAs for 72 h and incubated with human amylin as described previously.
4
Transient Silencing of AURKA and eIF4E
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Cells were seeded at 60% confluency in 10% FBS‐containing DMEM for 24 h in p60 plates. AURKA and eIF4E were transiently silenced by using siAURKA and sieIF4E (Invitrogen) for a total of 48 h. A negative siRNA control (Ambion, Austin, TX, USA) was used in each experiment. Transfection of cells was achieved by using a LipoJet reagent (SignaGen) according to the manufacturer's instructions. Following 24‐h transfection, medium was replaced with DMEM, supplemented with 5% FBS and antibiotics for another 24 h prior to harvesting. Validation of AURKA and eIF4E knockdown was assessed by qPCR and western blot analyses.
5
Microglial Phagocytosis Assay
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Reagents for reverse transcription (High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor) and quantitative PCR (Taqman Assays, TaqMan OpenArray Mouse Phagocytosis Panel, and TaqMan Fast Advanced Master Mix), red fluorescent microspheres (FMS), 2.0 μm, Hoechst 33342, CellMask Orange Actin Tracking Stain, CellMask Green Actin Tracking Stain, RPMI were obtained from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Opti-MEM was from Gibco, negative siRNA control was from Ambion, Lipofectamine was from Thermo Fisher Scientific, Inc., Aβ1–42 and Aβ1–42 HiLyte 488 were from AnaSpec, Inc. (Fremont, CA, USA). JQ1, GSK12101517, IBET-762, OTX-015, PFI-1, Cytochalasin D were obtained from Sigma-Aldrich (St. Louis, MO, USA). Heat-inactivated foetal bovine serum (FBS), Accutase solution, penicillin, streptomycin, L-glutamine, 3-(4,5-dimethyl-2-tiazolilo)-2,5-diphenyl-2H-tetrazolium bromide (MTT), propidium iodide, TRI-reagent, DNase I, dithiothreitol (DTT), anhydrous dimethyl sulfoxide (DMSO), lipopolysaccharide from Escherichia coli O55:B5 (toxicity 3,000,000 U/mg), and all other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).
6
Silencing AURKA and eIF4E in cells
7
Transfection of Cells with miR-24 Modulators
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Transfection of all different cell types was performed using Lipofectamine RNAiMAX (Invitrogen) reagent, mixed with anti-hsa-miR-24, pre-hsa-miR-24 or scrambled controls (Ambion). For each transfection, 120 pmol of anti- or pre-miR (final concentration at transfection was 50 nM) was diluted in 250 μl Opti-MEM (Gibco). Two hours after transfection, cells were treated with IL-6 as described above and harvested 24 h later. RNA was extracted using TRIzol (Invitrogen). Successful transfection (>80% of all cells) was confirmed by visual fluorescent microscopic analysis and FACS, sorting for Cy3-labelled scr-miR control. Some experiments utilized simultaneous transfection with 150 pmol siRNA (final concentration 50 nM) directed against CHI3L1/Chi3l1, with negative siRNA controls (Ambion).
8
Transfection and cytokine treatment
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