The target sequence [sense 5′-AAAGACAGCCTTAGCTTTGAG-3′] for human CIB1 RNAi was selected with the use of the siRNA target finder program of Ambion. The annealed oligonucleotides including sense and anti-sense strands of the target sequence were inserted into the pSUPER.retro vector (OligoEngine). As a control, annealed oligonucleotides containing a target sequence (sense 5′-GGCTACGTCCAGGAGCGCACC-3′) for a GFP shRNA were inserted into the same vector. The nucleotide sequences of the various inserts were confirmed by DNA sequencing. SH-SY5Y cells were transfected with pSUPER.retro vectors encoding either the control (GFP) or CIB1 shRNA, and stable transfectants were selected in the presence of puromycin (0.25 μg/ml). Rat CIB1 siRNA (sense 5′-AAGAGTCACTGCATACCCGAG-3′), Mouse ASK1 siRNA (sense 5′-AATTGCAGTCTGCACAGCCTTTCGG-3′), or control GFP siRNA (sense 5′-GGCTACGTCCAGGAGCGCACC-3′) oligonucleotides were obtained from Invitrogen and introduced into primary rat mesencephalic dopaminergic neurons by transfection for 48 h with the use of RNAiFect (Qiagen).
Rnaifect
Manufactured by Qiagen
Sourced in Germany, United States
RNAiFect is a transfection reagent designed for efficient delivery of small interfering RNA (siRNA) or other nucleic acids into mammalian cells. It facilitates the uptake of siRNA into cells, enabling RNA interference (RNAi) studies.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
Psuper retro vector, by Oligoengine (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Elx808 microplate reader, by Agilent Technologies (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (1 mentions)
Geneporter 3000, by Genlantis (1 mentions)
Ambion silencer select, by Thermo Fisher Scientific (1 mentions)
Plncx2, by Takara Bio (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Polybrene, by Merck Group (1 mentions)
Ab89911, by Abcam (1 mentions)
Mirvana microrna isolation kit, by Thermo Fisher Scientific (1 mentions)
Miscript reverse transcription kit, by Qiagen (1 mentions)
Ehuegfp, by Merck Group (1 mentions)
96 well plate, by Greiner (1 mentions)
Cy3 sirna, by Horizon Discovery (1 mentions)
Superfect transfection reagent, by Qiagen (1 mentions)
21 protocols using rnaifect
1
Silencing CIB1 and ASK1 in Neurons
2
Lovastatin Lactone Cytotoxicity Assays
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Cells were seeded in 24-well plates at a density of 1 × 105 cells per well (DNA fragmentation assays; Figure 8C, 8D ), in 6-well plates at a density of 2 × 105 cells per well (Western blot analyses, Figure 8A, 8B , lower panel) and in 96-well plates at a density of 5 × 103 cells per well (WST-1 tests; Figure 8A, 8B , upper panel), and were allowed to adhere for 2-3 h. Transfection was performed as described previously [26 (link), 28 (link), 29 (link)]. In brief, cells were transfected with an equal ratio (w/v) of RNA to transfection reagent for 24 h in 10% DMEM prior to incubation with lovastatin lactone. Subsequently, cells were washed with PBS, transfected again in serum-free DMEM to provide constant transfection conditions, and incubation with vehicle or lovastatin lactone was started. Transfections were carried out using RNAiFect® as transfection reagent (Qiagen, Hilden, Germany). SiRNA was obtained from Qiagen. The nonsilencing negative control RNA was from Eurogentec (Cologne, Germany). Final concentrations of COX-2 SiRNA and non-silencing SiRNA were 2.5 μg/ml, respectively.
3
Culturing and Transfecting Dopaminergic Neuroblastoma Cells
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Human dopaminergic neuroblastoma SH-SY5Y cells were routinely maintained under a humidified atmosphere of 5% CO2 at 37 °C in DMEM (Invitrogen) supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml). Transfection of cells was performed with Lipofectamine2000 (Invitrogen) for SH-SY5Y cells or RNAiFect (Qiagen) for primary mesencephalic neuronal cells.
4
Transient GP96 Expression Optimization
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Transfections were performed using Lipofectamine 2000 (Invitrogen) and RNAiFect (Qiagen) following manufacturer's instructions. GP96 ON-TARGETplus smartpools siRNA were purchased from Dharmacon (Thermo Fisher Scientific, Europe). Mammalian expression vectors used for transient GP96 expression were described previously [15] (link).
5
Cell Viability Assay with MTS
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Cell proliferation and viability was assessed using the CellTiter 96® AQueous One solution cell proliferation assay (MTS; Promega GmbH, Mannheim, Germany). This assay contains a tetrazolium compound (MTS) and an electron coupling reagent, phenazine ethosulfate (PES). MTS can be bioreduced by cells into formazan, which is soluble in cell culture medium. The quantity of the formazan product as measured spectrophotometrically is directly proportional to the number of viable cells. The assay measures dehydrogenase enzyme activity found in metabolically active cells. MCF-7 or MDA-MB-231 cells were seeded in 96-well plates at a density of 1–3×104 cells per well in 100 µl RPMI 1640 medium, and incubated overnight at 37°C. The cells were washed 2 times with PBS then different concentration (10, 25, 50,75, 100, 250, 500, 1000 nM) of siRNAs were mixed with 1.5 µl RNAifect (Qiagen GmbH) and added at a final volume of 100 µl per well. After 48 h, the Aqueous One Solution containing MTS/PES was added and the optical density was measured at 490 nm using an ELx808 microplate reader (BioTek Instruments GmbH, Bad Friedrichshall, Germany) after 6 h. This experiment was performed in triplicate wells. The absorbance measured from control siRNA (siNON) treatment was normalized to 100% viability.
6
Overexpression and Silencing of IRF7 in HCEn Cells
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HCEn cells were transfected with an IRF7-expressing plasmids using a CMV promoter (pCMV6 backbone, OriGene, Rockville, MD) with gene porter 3000 (Genlantis, San Diego, CA). The extracted proteins were confirmed by western blot analyses using an anti-IRF7 antibody (Santa Cruz Biotechnology, Dallas, TX) and HRP-conjugated secondary antibody (Cell Signaling, Danvers, MA).
For siRNA transfection, HCEn cells were transfected with IRF7 siRNA (Santa Cruz Biotechnology, Dallas, TX) using RNAifect (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
For siRNA transfection, HCEn cells were transfected with IRF7 siRNA (Santa Cruz Biotechnology, Dallas, TX) using RNAifect (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
7
Transfection of E13 Epithelia
8
siRNA Knockdown of Cytoskeletal Regulators
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The following duplexed siRNAs (predesigned Ambion Silencer Select) were obtained from Life Technologies: LIMK 1, 156129 and s69232; LIMK 2, 156133 and s69236; TPPP/p25, s91408 and s91407; nonmuscle cofilin, Cfl1, s63902; cdc42 siRNA, s63741; and negative control siRNA # 1. SMGs were transfected with 500 nM siRNA using RNAiFect (Qiagen, Valencia, CA) and cultured for 24–48 h, as we reported previously (Daley et al., 2009 (link))
9
FAP-α Depletion via RNA Interference
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Depletion of FAP-α was performed with two siRNA sequences (SI00064050; SI00064057, 1 μg of each) and 6 μl RNAiFect (all from Qiagen, Germantown, MD, USA). Optimal transfection conditions were determined by Alexa Fluor 555 labelled non-silencing siRNA, with a scrambled sequence, which was also used as negative control. siRNA targeting Lamin A/C (SI03650332) served as positive control.
10
Lamin A Variant Expression Vectors
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The expression vector pcDNA™5/FRT/TO (Invitrogen) was cut with Bam H I and Xho I. Fragments of lamin A, progerin, lamin A R133L, or lamin A L140R were ligated into the corresponding restriction sites of pcDNA™5/FRT/TO. The resulting expression vectors were transfected into HEK293 cells by using Fugene. We created vectors based on pLNCX2 (Clontech, Palo Alto, CA, USA) that expressed wild-type lamin A or progerin for retroviral infection, which was done as described previously [26 (link)]. Briefly, VSMCs or HUVECs (passages 4-6) were plated at 5×105 cells in 100-mm dishes at 24 hours before infection. Then the culture medium was replaced by retroviral stock medium supplemented with 8 μg ml−1 polybrene (Sigma). From 48 hours after infection, the cells were selected by culture for 7 days in 500 μg ml−1 G418. After selection, 2×105 cells were seeded in 100-mm dishes on the 8th day post-infection, which was designated as day 0. The respective empty vectors were used as controls. In some experiments, after retroviral infection had been performed, siRNAs purchased from Ambion or Invitrogen were transfected at 10 nmol l−1 with RNAiFect (Qiagen) or Lipofectamine RNAiMAX (Invitrogen) according to the instructions of the respective manufacturers.
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