P3-P5 BMSCs were inoculated into a six-well plate (1×105 cells/well) containing fetal bovine serum (FBS, Gibco, US) medium. When the cells reached a confluency of 30–50%, the mixture of miR-31agomir (RIBBIO Guangzhou, China) and transfection reagent (Polyplus, France) and transfection reagent were separately added to the wells and the cells were further cultured for 12 h before the medium was replaced with normal medium. The final concentrations for miR-31agomir and miR-31antagomir were 50ηM and 100ηM, respectively.
Transfection reagent
Manufactured by Polyplus Transfection
Sourced in France, United States, China
Transfection reagent is a laboratory product used to introduce genetic material, such as DNA or RNA, into cells. It facilitates the uptake and expression of the introduced genetic material within the target cells. The transfection reagent provides a method for efficient delivery of genetic payloads into cells.
Automatically generated - may contain errors
14 protocols using transfection reagent
1
miR-31 transfection in BMSCs
2
Generating Luciferase-Expressing B16-F10 Cells
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The vector containing CMV-luciferase2 vector (pGL4.50[luc2/CMV]) (Promega, Madison, WI, USA) and transfection reagent (Polyplus transfection, France) were prepared in advance. B16-F10 cells were seeded in 6 cm plate one day before transfection. Cells density is around 70% during transfection procedure. The jetPEITM reagent (10 µL) dissolved in 250 µL of NaCl buffer was then added into DNA buffer (5 μg plasmids with 250 µL of NaCl buffer), the mixture was then incubated at 25 °C for 25 min. The mixture was finally added to the B16-F10 cells for an incubation period of 1 day. Luc2 expression cells were selected by hygromycin B 200 μg/mL for another two weeks and named as B16-F10/luc2 cells [18 (link)].
3
Inducing Allergic Airway Inflammation in Mice
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To establish the HDM-induced allergic airway inflammation model, mice were anesthetized with isoflurane before intranasal aspiration of 40 μl of lyophilized HDM extract solution (0.25 μg/μl; Greer Laboratories) on days 0, 1, and 2 and then days 8 to 12. On days 8, 10, and 12, mice were transfected intranasally with 20 μg of siRNA dissolved with transfection reagent (Polyplus-transfection, France) 2 h before the HDM challenge. The control group was sensitized and challenged with phosphate-buffered saline (PBS) alone. All mice were anesthetized with avertin (25 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) and sacrificed for analysis 48 h after the last HDM challenge. The sequence of SPRR3 siRNA was as follows: forward 5′-GAGCCAGAUUAUACUACAATT-3′, reverse 5′-UUGUAGUAUAAUCUGGCUCTT-3′.
4
Silencing MIF in Rheumatoid Arthritis FLSs
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The negative control (NC) siRNA and siRNA targeting macrophage migration inhibitory factor (MIF) were synthesized by Ruibo Co., Ltd. (Guangzhou, China). siRNA targeting MIF or NC siRNA was transfected into RA FLSs using transfection reagent (PolyPlus Transfection, Strasbourg, France) according to the manufacturer’s instructions. Total RNA was extracted by TRIzol to conduct RT-qPCR or cDNA library construction for RNA transcriptome sequencing by LC-BIO Technologies Co., Ltd. (Hangzhou, China).
5
Targeting SOX2 and OCT4 in TNBC Cells
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The TNBC cell lines MDA-MB-231 and HCC-1937 were obtained from the Cell Bank of the Representative Culture Preservation Committee of the Chinese Academy of Sciences. All the cells were cultured in DMEM supplemented with 10% FBS (HyClone; Cytiva) at 37°C in 5% CO2 and subcultured every 2–3 days. siRNA or negative control RNA were obtained from Shanghai GenePharma Co. Ltd. The cells were seeded in six-well plates at a density of 3×105 cells/well. The transfections were performed using Transfection Reagent (Polyplus Transfection SA) according to the manufacturer's instructions. The transfection efficiency was confirmed via quantitative PCR. For the spheroid formation assay, the cells were transfected with siRNA targeting SOX2 or OCT4 for 24 h prior to treatment with bufalin (0.5 µM).
6
Silencing nNav1.5 Impacts Glutamate Levels
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Knockdown was conducted in order to investigate the effects of silencing nNav1.5 expression in MDA-MB-231 cells (which expressed the most significant nNav1.5 mRNA upregulation) on the concentration of glutamate. SiRNA on MDA-MB-231 cells were produced via transient transfection, whereby the siRNA sequences against nNav1.5 and controls were acquired commercially (SMARTpool siRNA reagents from Dharmacon). 3 × 104 cells of MDA were seeded in a well of 24-well plate. The cells were incubated overnight. Before starting the treatment on the next day, working concentration of siRNA from a 1 µM stock resuspension was prepared/ by adding 6 µl of it into 44 µl of serum free media in a tube. A transfection solution was prepared by adding 3 µl of transfection reagent (Polyplus-transfection SA, France) into 47 µl of serum free media in another tube. Both tubes were slightly vortexed. The transfection solution was later added into the siRNA suspension and was vortexed slightly and then left at room temperature for 5 min. The cells that were incubated in the wells overnight was removed of old media 500 µl of new media added. The siRNA transfection solution was then added into the well and incubated at 37 °C for 5 h before the media was changed. To confirm the success of nNav1.5 knockdown, the gene in expression in the knock downed cells were measured using qPCR.
7
Knockdown of GSK-3β, Rho-T1, Dynactin1, and P62
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The siRNA specific for GSK-3β, Rho-T1, Dynactin1, and P62 was synthesized by RiboBio Corporation (Guangzhou, China). AML12 cells were seeded in 6-well or 24-well plates. At 40% confluence, the cells were cultured in fresh medium without penicillin and streptomycin, together with transfection reagent (Polyplustransfection, Illkirch, France) and 20 nM GSK-3β, Rho-T1, Dynactin1,and P62 siRNA or negative control siRNA for 24 h, according to the manufacturer’s protocols. Then, the cells were subjected to other treatments as required by the experiments.
8
Silencing Key Transcriptional Regulators
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To silence the expression of YAP, LATS1, MST1, TEAD1, TEAD2, TEAD3 and TEAD4, all siRNAs as well as the non-targeting control siRNA were purchased from Gene Pharma (Shanghai, China) and transfected using the Transfection Reagent (Polyplus, NY, USA) according to the manufacturer's protocol. For each gene, two individual siRNAs were used (S1 Table ).
9
Tumor Suppression via miR-424-5p
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5-week-old immunodeficient male BALB/c athymic nude mice (Huafukang Biotechnology Ltd, Beijing, China) were bred in aseptic conditions and maintained in Shandong University Experimental Animal Center. All mice were subcutaneously injected with SMMC7721 HCC cells (1 × 107 cells) in the left flank region. When visible tumor appeared, the mice were randomly divided into miR-424-5p transfection group and mock control group. Tumors were injected with 10 μg of pCMV-miR-424-5p plasmid (Origene Technologies, Rockville, MD) or empty pCMV vector control in 50 μl transfection reagent (Polyplus-transfection Inc, New York, USA) according to the manufacturer's instructions. This injection was performed once every 3 days for a total of 12 days before the mice were sacrificed and the tumors were isolated. The tumor volume (V) was obtained by measuring the length (L) and width (W) with a caliper and calculated with the formula: Volume (mm3) = [width2 (mm2) × length (mm)]/2. Mice were maintained in accordance with guidelines of the Institutional Animal Care and Use Committee, and all the animal studies performed were approved Shandong University Institutional Animal Care and Use Committee.
10
Validation of RGMA 3'UTR-miR-552-3p Interaction
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A 1,000-bp fragment of the RGMA 3'-UTR containing miR-552-3p-binding sites was inserted into a luciferase reporter plasmid (OBIO technology, Shanghai, China), and a synthetic RGMA 3'-UTR mutant fragment was inserted into an equivalent reporter plasmid. For the luciferase assays, HEK-293T (ATCC, Cat# CRL-3216, RRID:CVCL_0063) cells were seeded in 6-well plates in triplicate and allowed to settle for 24 h. The cells were then co-transfected with 10 ng of firefly luciferase reporter plasmid and an equal amount (50 pmol) of miR-552-3p mimics or scrambled negative control RNA using transfection reagent (Polyplus Transfection, USA). Forty-eight hours after transfection, the cells were assayed using a luciferase assay kit (Promega, Madison, WI, USA).
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