siRNA transfection was performed with siRNA reagent system (Santa Cruz, sc-45064) following the manufacturer’s instructions. Briefly, cells were replated in six-well plates to 40% confluence in antibiotic-free 10% FBS-containing medium. For each transfection, 6 μl of WT1 siRNA (Santa Cruz, sc-36846), TAZ siRNA (Thermo Fisher, 122501), and unconjugated control siRNA-A (Santa Cruz, sc-37007) in 100 μl of transfection medium (Santa Cruz, sc-36868) were mixed with 6 μl of transfection reagent (sc-29528) in another 100 μl of transfection medium. The mixtures were incubated for 30 min at room temperature. Each well was washed with 2 ml of transfection medium once and then filled with a prepared reagent mixture plus 0.8 ml of transfection medium. After incubation overnight, an additional 1 ml of medium supplemented with 20% FBS with 2% antibiotics was added to each well for another 24 h. The transfection mixture was removed and replaced with normal growth medium on the following day. All experimental measurements were performed 24 h following replacement of the medium. The sequence information of siRNAs will be available upon request.
Sirna reagent system
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The SiRNA reagent system is a laboratory tool designed for the delivery of small interfering RNA (siRNA) into cells. It provides the necessary components for efficient siRNA transfection, which is a widely used technique for gene expression silencing in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Sc 37007, by Santa Cruz Biotechnology (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Control sirna, by Thermo Fisher Scientific (2 mentions)
Anti gapdh sirna, by Thermo Fisher Scientific (2 mentions)
Sc 37265, by Santa Cruz Biotechnology (2 mentions)
Sc 41113, by Santa Cruz Biotechnology (2 mentions)
Control sirna, by Santa Cruz Biotechnology (2 mentions)
Taz sirna, by Thermo Fisher Scientific (1 mentions)
Plenti6 v5 dest, by Thermo Fisher Scientific (1 mentions)
Transfection reagent, by Santa Cruz Biotechnology (1 mentions)
Scramble sirna, by Santa Cruz Biotechnology (1 mentions)
Taqman reverse transcription reagent, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Sc 41446, by Santa Cruz Biotechnology (1 mentions)
Gapdh sirna, by Thermo Fisher Scientific (1 mentions)
Am17110, by Thermo Fisher Scientific (1 mentions)
Mir 200a, by Thermo Fisher Scientific (1 mentions)
Mir 101, by Thermo Fisher Scientific (1 mentions)
Fluorescent secondary antibody, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
30 protocols using sirna reagent system
1
siRNA Transfection for Gene Silencing
2
Silencing CTCF and TP53 in LFS Cell Line
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Briefly, CTCF, TP53 (Santa Cruz; sc-35124 and sc-44218) and non-silencing control as well as fluorescein-conjugate (Santa Cruz, sc-37007 and sc-36869) were used at 60 nM to transfect LFS cell line, 3335, using siRNA Reagent System (Santa Cruz, sc-45064) in serum-free media for 6h according to manufacturer's instructions, Knockdown and transfection efficiency of siRNAs were confirmed by RT-PCR and Fluorescence Microscopy. The primers used for CTCF and TP53 amplifications were as follows: forward/ 5′-GAA ATG GAA GGT GAT GCA GTC GAA GC -3′, reverse/ 5′- CCG GTC CAT CAT GCT GAG GAT CA -3′; and : forward/ 5′- GCC ATG GAG GAG CCG CAG TCA-3′, reverse/ 5′-TCA GTC TGA GTC AGG CCC TTC TGT CTT-3′.
3
Overexpression of CREB1 in Glioma Cells
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The miR-138 mimics and miRNA-negative control (NC) mimics were obtained from Qiagen GmbH. U87 and U251 cells were transfected with miR-138 mimics or miRNA-NC by Lipofectamine™ 2000 (Thermo Fisher Scientific, Inc.). The target sequences of miR-138 mimics were as previously described (19 (link)). The scrambled sequence (5′-UUCUCCGAACGUGUCACGUTT-3′) was used to create non-targeting miR-NC and GFP-siRNA. CREB1 siRNA (sc-35111) and siRNA Reagent System (sc-45064) were purchased from Santa Cruz Biotechnology, Inc. and all transfections were conducted according to the manufacturer's instructions. CREB1 overexpression vectors were purchased from GenePharma and successfully transfected into the corresponding cells according to the manufacturer's instructions in the presence of Lipofectamine™ 2000 (Thermo Fisher Scientific, Inc.). A full-length human CREB1 complementary DNA containing the entire coding sequence tagged with GFP or GFP alone (Lenti-GFP) was cloned into the lentiviral vector pLenti6/V5-DEST (Invitrogen; Thermo Fisher Scientific, Inc.) to create the complete functional overexpression vector Lenti-CREB1. The efficacy of the transfection was tested by using western blotting.
4
Silencing Nox2 Gene Expression
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siRNA against gp91phox (sc-35503), related RT-PCR Primer (sc-35503-PR), Control siRNA (sc-37007), and siRNA Reagent System (sc-45064) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); transfections were performed by using the methods and reagents described by the manufacturer.
5
Knockdown of TAK1 in BJAB Cells
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Small interfering RNA (siRNA) Reagent System and a pool of 4 target-specific siRNA against human TAK1 was purchased from Santa Cruz Biotechnology. BJAB cells were transfected according to the manufacturer's instructions. After 48 and 72 hours cells were activated with 2.5 µg/ml anti-Ig and 1 µg/ml CpG for 30 min and subjected to Western blot analysis.
6
Silencing TRPM7 in Vascular Smooth Muscle Cells
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A small interfering RNA (siRNA) was generated against TRPM7. siRNA for knocking down TRPM7 was synthesized by Qiagen (USA). The DNA target sequence of the annealed double strand siRNA that we used was: 5′-CCT GTA AGA TCT ATC GTT CAA-3′ . siRNA, with a nonsilencing oligonucleotide sequence (nonsilencing siRNA) that does not recognize any known homology to mammalian genes, was also generated as a negative control.
Cells (VSMCs) were seeded at a density of 2×105 cells/well in 6-well plates and grown in DMEM containing 15% FBS and antibiotics. One day after seeding, cells were transfected with siRNA using siRNA Reagent System (Santa Cruz Biotechnology Inc, USA) according to the manufacturer's instructions. Briefly, siRNA (40 pmol) was mixed with transfection medium (100 µL) to which transfection reagent (6 µL) plus transfection medium (100 µL) was added. After mixing (45 minutes), the formulation was added dropwise onto the cells. Control cells were exposed to transfectant in the absence of siRNA. 18 hours after transfection, media was changed to DMEM containing 15% FBS and antibiotics. Cells were lysed after 24 hours, and gene silencing was monitored at the mRNA level by RT-PCR.
Cells (VSMCs) were seeded at a density of 2×105 cells/well in 6-well plates and grown in DMEM containing 15% FBS and antibiotics. One day after seeding, cells were transfected with siRNA using siRNA Reagent System (Santa Cruz Biotechnology Inc, USA) according to the manufacturer's instructions. Briefly, siRNA (40 pmol) was mixed with transfection medium (100 µL) to which transfection reagent (6 µL) plus transfection medium (100 µL) was added. After mixing (45 minutes), the formulation was added dropwise onto the cells. Control cells were exposed to transfectant in the absence of siRNA. 18 hours after transfection, media was changed to DMEM containing 15% FBS and antibiotics. Cells were lysed after 24 hours, and gene silencing was monitored at the mRNA level by RT-PCR.
7
AhR Gene Silencing in Human PBMCs
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Specific siRNA targeting AhR (Santa Cruz) was used to inhibit gene expression in human PBMC. After one day of cell culture in serum free media, the following processes for transfection were performed using a siRNA reagent system (Santa Cruz). Mixed siRNA for AhR and transfection reagent were reacted for 45 minutes at room temperature, diluted in transfection media, and applied to cells without culture media. After five hours in a CO2 incubator at 37℃, samples were cultured for one day in media containing FBS and a twofold greater quantity of antibiotics than previously added. Thereafter, culture media was removed, replenished with new media, and cultured for an additional day.
8
Downregulation of APE1 by siRNA
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Two siRNAs against APE1 and negative control siRNA were transfected using siRNA Reagent System (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) according to the manufacturer’s instructions (siRNA Transfection Protocol). The down-regulation of APE1 gene expression was analyzed by immunofluorescence staining.
9
Macrophage Interaction with Candida albicans
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Human monocyte-derived macrophages were treated with P17 (200 µg/ml) for 24 h and were allowed to interact for 40 min at 37°C with C. albicans blastospores (at a ratio of 0.3 yeast per macrophage) as previously described (36 (link), 40 (link)). Unbound yeasts were removed by four washes with medium. h-MDMs were then incubated at 37°C for 4 h. After incubation, the medium was removed and cells were lysed. The CFU of C. albicans were quantified after plating on Sabouraud plates for 24–48 h at 37°C.
In some experiments, monocyte-derived macrophages were incubated with 1 µg per well of MR siRNA (Santa Cruz Biotechnology sc-45360) and/or Dectin-1 siRNA (Santa Cruz Biotechnology sc-63276) for 6 h into siRNA transfection medium (siRNA reagent system, Santa Cruz Biotechnology sc-45064) according to the manufacturer’s instructions before the addition of P17.
In some experiments, monocyte-derived macrophages were incubated with 1 µg per well of MR siRNA (Santa Cruz Biotechnology sc-45360) and/or Dectin-1 siRNA (Santa Cruz Biotechnology sc-63276) for 6 h into siRNA transfection medium (siRNA reagent system, Santa Cruz Biotechnology sc-45064) according to the manufacturer’s instructions before the addition of P17.
10
siRNA Transfection Optimization for Cell Lines
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siRNA transfection was performed with the siRNA reagent system (sc-45064, Santa Cruz Biotechnology) following the manufacturer‘s instructions. Briefly, 2 × 105 cells/well were replanted in six-well plates in antibiotic-free medium overnight or until they reached 80% confluence. For each transfection, 6 µL of HSP27 siRNA or unconjugated control siRNA-A in 100 µL of transfection medium was mixed with 6 µL of transfection reagent (all materials from Santa Cruz Biotechnology) in another 100 µL of transfection medium. The mixtures were incubated for 30 min at room temperature. Each well was washed with 2 mL transfection medium. After incubation overnight, 1 mL of medium supplemented with 20% FBS with 2% antibiotics was added to each well for another 24 h. The transfection mixture was removed and replaced with normal growth medium on the following day. All experimental measurements were performed 24 h after medium replacement.
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