The extraction socket was treated with 2 µL of mixture that comprise Pluronic® F-127 (Sigma-Aldrich, Steinheim, Germany, cat no. CAS: 9003-11-6), 0.05% dimethyl sulfoxide (DMSO; Duchefa Biochemie, Haarlem, The Netherlands, cat no. CAS: 67-68-5), siTran transfection reagent (Origene Technologies Inc. Rockville, MD, USA cat no. TT30001), and scrambled siRNA (negative control) (Origene Technologies Inc. Rockville, MD, USA, cat no. SR30004) for control groups and 2 µL mixture of Meox2 siRNA (Origene Technologies Inc. Rockville, MD, USA, cat no. SR408862), siTran transfection reagent, Pluronic® F-127, and 0.05% DMSO for the siRNA therapy experimental group. The final concentration of siRNA in the mixture was maintained at 100 nM. To deliver the chemical into the socket, gas-tight Hamilton syringe 84877 (Hamilton, Reno, NV, USA) was used. The area was sealed using fibrin sealant (Tisseel; Baxter, Deerfield, IL, USA, cat no. SKU 1506079) after the delivery of treatment material into the socket in order to ensure that the treatment material is retained in the socket.
Sitran transfection reagent
Manufactured by OriGene
Sourced in United States
SiTran is a transfection reagent developed by OriGene. It is designed to facilitate the delivery of nucleic acids, such as plasmid DNA or siRNA, into mammalian cells for various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green qpcr supermix udg, by Thermo Fisher Scientific (1 mentions)
M mlv reverse transcription system, by Promega (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Small interfering rnas sirnas, by Thermo Fisher Scientific (1 mentions)
Ecl western blotting detection reagent, by Cytiva (1 mentions)
Peroxidase envision kit, by Agilent Technologies (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Gotaq qpcr master mix, by Promega (1 mentions)
Bca protein assay kit, by Keygen Biotech (1 mentions)
Sirna duplexes, by Horizon Discovery (1 mentions)
Sirna duplexes, by OriGene (1 mentions)
Scrambled sirna, by OriGene (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Trilencer 27 human sirna, by OriGene (1 mentions)
Pluronic f 127, by Merck Group (1 mentions)
Tisseel, by Baxter (1 mentions)
9 protocols using sitran transfection reagent
1
siRNA Delivery to Surgical Socket
2
Molecular Assays for DNA Repair Pathway
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RPMI 1640, fetal bovine serum (FBS), TRIzol® reagent, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT), and the platinum SYBR Green qPCR SuperMix-UDG were purchased from Invitrogen (Carlsbad, CA, USA). M-MLV Reverse transcription system and GoTaq® qPCR Master Mix were from Promega (Madison, WI, USA). BCA Protein Assay Kit was from KeyGEN Biotech (Nanjing, China). siTRAN transfection reagent was from OriGene (Beijing, China). Primers and small interfering RNAs (siRNAs) were synthesized respectively by Invitrogen (Shanghai, China) or GenePharma (Suzhou, China). Anti-XPA (sc-853), anti-GAPDH antibodies and HRP-conjugated secondary antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), ECL Western blotting detection reagents was from Amersham Biosciences (Piscataway, NJ, USA). Peroxidase Envision Kit was from Dako (Carpinteria, CA, USA). Cisplatin and all other reagents were of molecular biology grade and obtained from Sigma-Aldrich (Shanghai, China).
3
Silencing Ferritin and RapGEF2 in Cells
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Small short interfering RNA (siRNA) was used to transiently silence Ferritin heavy chain 1 (FTH1) and RAPGEF2. To decrease the expression of Ferritin, a pool of three siRNA duplexes (cat# SR301663, Origene Technologies, Rockville, MD) against FTH1 and scramble siRNA (cat# SR30004) were used. To reduce the expression of RapGEF2, a pool of four siRNA duplexes (cat# L-009742-00-0005, Dharmacon, Lafayette, CO) was applied. siRNA were delivered to cells by SiTran Transfection reagent (Origene Technologies). After transfection for 72 hr, cells were treated with cAMP for intracellular labile Fe(II) measurement. Cells in a subset of wells were harvested for RNA and protein extraction and subsequent qRT-PCR and immunoblot assays.
4
siRNA Depletion of Clk1, 2, and 4
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SiRNAs that targeted separate areas of mRNA were pretested and the most efficient were used to deplete Clk1,2 and 4. Clk1 siRNAs (IDs: SR300856C), Clk2 siRNAs (IDs:SR416008A‐C) and Clk4 siRNAs (IDs: SR408785A‐C) along with scrambled control siRNA was purchased from Origene, and transfected using Origene's siTran transfection reagent. Cells were treated with the siRNA + Origene siTran transfection agent on Day 2 of differentiation. SiRNA was re‐transfected with every media change until the cells were harvested for RNA.
5
Hsp60 siRNA Knockdown in MM Cells
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Hsp60 (HSPD1) human siRNA oligo duplex kit (SR302265; OriGene) containing three separate siRNA constructs and siTRAN transfection reagent (TT320002; OriGene) were used to transfect MM cells as per manufacturer’s instructions. Universal scrambled negative control siRNA duplex (SR30004; OriGene) was transfected into MM cells in the same manner. Knockdown efficiency was measured through immunoblotting as described above.
6
Silencing XBP-1 Protects Cells from Oxygen-Glucose Deprivation
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We followed the methods of Bai et al. [24 (link)]. XBP-1 siRNA and negative control siRNA were chemically synthesized by Shanghai GeneChem Co., Ltd. (Shanghai, China). The sequences were as follows: XBP-1 siRNA, sense: 5′-CACCGGCTGCTCCAGCTCGCTCATC-3′; antisense: 5′-AAACGATGAGCGAGCTGGAGCAGCC-3′ and negative control siRNA, sense: 5′-UUCUCCGAACGUGUCACGUTT-3′; antisense: 5′-ACGUGACACGUUCGGAGAATT-3′.
HT22 cells and C8-B4 cells were seeded in 6-well plate at a density of 4 × 104 per well. The contents of 0.33 μg siRNA and 5 μl siTran transfection reagent (Origene, MD, USA) per well were diluted separately in serum free Opti MEM for a final volume of 250 μl, gently mixed, and incubated for 5 min at room temperature. Then, the diluted siRNA solution and the diluted siTran transfection reagent were mixed gently and incubated for 20 min at room temperature. The diluted siRNA/siTran transfection reagent complex was added to the plates. After transfection with siRNA for 24 h, the cells were exposed to OGD/R and then harvested for assay.
HT22 cells and C8-B4 cells were seeded in 6-well plate at a density of 4 × 104 per well. The contents of 0.33 μg siRNA and 5 μl siTran transfection reagent (Origene, MD, USA) per well were diluted separately in serum free Opti MEM for a final volume of 250 μl, gently mixed, and incubated for 5 min at room temperature. Then, the diluted siRNA solution and the diluted siTran transfection reagent were mixed gently and incubated for 20 min at room temperature. The diluted siRNA/siTran transfection reagent complex was added to the plates. After transfection with siRNA for 24 h, the cells were exposed to OGD/R and then harvested for assay.
7
Embryonic Tooth Germ Cultivation and Transplantation
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The embryonic mice molar tooth germs at E14 were cultivated for 1 and 2 days, and transplanted into the kidney capsule, as described by [37 (link),49 (link)]. During cultivation, embryonic tooth specimens were transfected with scrambled siRNA (negative control) (Cat# SR30004, Origene Technologies Inc., Rockville, MD, USA), Fubp1 siRNA (Cat# SR418703, Origene Technologies Inc., Rockville, MD, USA) using siTran transfection reagent (Cat# TT30001, Origene Technologies Inc., Rockville, MD, USA), in separate culture dishes for 1 day in Opti-MEM (Cat# 31985-070, Gibco, Grand Island, NY, USA). The siRNAs were used at a final concentration of 100 nM. The naive and scrambled control did not show any differences in the expression of the gene (Data not shown).
8
Quantifying miRNA Regulation of THSD4
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To explore the regulation of gene expression by miRNAs, it is necessary to identify the target genes and regulatory effects of each miRNA. In order to further demonstrate the regulatory role of THSD4, human embryonic kidney 293T (HEK293T) cells (American Type Culture Collection, Manassas, VA, USA) were transfected with short RNA sequences using siTran transfection reagent (Origene) according to the manufacturer’s protocol. Cells were then incubated for 48 h, and the expression level of THSD4 mRNA was quantified by real-time quantitative polymerase chain reaction (RT-qPCR). The fold change in mRNA was computed based on the change in threshold cycle (Ct) and represented by 2−ΔΔCt values.
9
Reducing SCD1 Expression via siRNA
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RNA interference to reduce SCD1 expression was performed with a set of three siRNA oligonucleotides (Trilencer-27 human siRNA) obtained from Origene Technologies Inc. (Rockville, MD, USA) (SR304248). The trilencer-27 universal scrambled negative control siRNA duplex (SR30004, OriGene) was used as the scrambled siRNA control (mock). The HepG2 cells were transfected for 24 h with increasing concentrations (0.1 nM, 1 nM and 10 nM) of each siRNA alone or in combination (SR304248A (1), SR304248B (2) and SR304248C (3)) using the siTRAN transfection reagent (Origene) following the manufacturer's protocol.
The total cell mRNA and the protein from these transfected cells were obtained and analyzed for SCD1 knockdown by real-time RT-PCR and Western blotting as previously described.
The total cell mRNA and the protein from these transfected cells were obtained and analyzed for SCD1 knockdown by real-time RT-PCR and Western blotting as previously described.
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