For the BCAR4 luciferase reporter assay, pRL-TK-BCAR4 or pRL-TK-BCAR4 mutant vectors and pGL3 control (Promega, Madison, WI) were transfected into HEK-293T cells along with miR-644a mimic or miR-644a inhibitor using ExFect® Transfection reagent (Vazyme) according to the instructions of the manufacturer. For miR-644a target gene CCR7 luciferase reporter assay, pRL-TK-CCR7-3΄UTR or pRL-TK- CCR7-3΄UTR mutant vectors and pGL3 control (Promega) were transfected into HEK-293T cells along with miR-644a mimic or miR-644a inhibitor using ExFect® Transfection reagent (Vazyme). The luciferase activity was measured 48 h after transfection using the Dual-Glo luciferase reporter assay system (Promega) according to the guidelines of the manufacturer. Luminescence values were recorded by a Victor X2 multilabel reader (PerkinElmer).
Exfect transfection reagent
Manufactured by Vazyme
Sourced in China
ExFect Transfection Reagent is a cationic lipid-based reagent designed for efficient and gentle delivery of nucleic acids into a variety of cell types. It facilitates the uptake of plasmid DNA, siRNA, and other nucleic acid molecules into cells for transfection studies.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (4 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (3 mentions)
Dual luciferase reporter assay system, by Promega (3 mentions)
Passive lysis buffer (plb), by Promega (3 mentions)
Dual luciferase system, by Promega (3 mentions)
Glomax 20 20 luminometer, by Promega (3 mentions)
Victor x2 multilabel reader, by PerkinElmer (2 mentions)
Dual glo luciferase reporter assay system, by Promega (2 mentions)
Polybrene, by Merck Group (2 mentions)
Puromycin, by Merck Group (2 mentions)
Pgl3 control, by Promega (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Transdetect pcr mycoplasma detection kit, by Transgene (2 mentions)
Fetal bovine serum (fbs), by Sartorius (2 mentions)
Pmir report luciferase reporter plasmid, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Enspire reader, by PerkinElmer (1 mentions)
Psicheck 2 luciferase vector, by Promega (1 mentions)
Dual luciferase assay system, by Meilun (1 mentions)
Ab181602, by Abcam (1 mentions)
93 protocols using exfect transfection reagent
1
Luciferase Assays for miR-644a Targets
2
Validating miR-15b-5p Regulation of ACSS2
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TargetScan software (version 7.0; targetscan.org/ ) (18 (link)) was used to identify the miR-15b-5p binding site in the 3'-UTR of ACSS2. The wild-type (WT) and mutant (MUT) 3'-UTR sequences of ACSS2 at the miR-15b binding site were chemically synthesized by General Biosystems, Inc., and inserted into the XhoI/NotI site of psiCHECK-2 luciferase vector (Promega Corporation) to construct the recombinant plasmids psiCHECK2-ACSS2-WT and psiCHECK2-ACSS2-MUT, respectively. miR-15b-5p mimics or miR-15b-5p mimic NC were co-transfected with psiCHECK2-ACSS2-WT and psiCHECK2-ACSS2-MUT into HEK-293T cells using the Exfect Transfection reagent (Vazyme Biotech Co., Ltd.). After 48 h, firefly and Renilla luciferase activities were measured on an EnSpire reader (PerkinElmer, Inc.) using the Dual Luciferase Assay System (Dalian Meilun Biology Technology Co., Ltd.).
3
Regulation of ACSS2 by miR-15b in VSMCs
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Empty pcDNA3.1 vector, ACSS2 overexpression plasmid (pcDNA3.1/ACSS2), miR-15b mimics/inhibitors and mimic/inhibitor negative controls (NC) were obtained from RiboBio Co., Ltd.. At 24 h before the transient transfections, an appropriate number of human aortic VSMCs were seeded into six-well plates with 2 ml of SMCM and grown to 60-70% confluency at 37˚C in an atmosphere containing 5% CO2. Then, the aortic VSMCs were divided into six groups, and miR-15b-5p mimic NC (5'-UCACAACCUCCUAGAAAGAGUAGA-3'; double stranded; 100 nM), miR-15b-5p mimic (5'-UAGCAGCACAUCAUGGUUUACA-3'; double stranded; 100 nM), miR-15b-5p inhibitor NC (5'-UCUACUCUUUCUAGGAGGUUGUGA-3'; single stranded; 200 nM), miR-15b-5p inhibitor (5'-AUCGUCGUGUAGUACCAAAUGU-3'; single stranded; 200 nM), pcDNA3.1 plasmid (4 µg/well), pcDNA3.1/ACSS2 plasmid (4 µg/well), miR-15b-5p mimics+pcDNA3.1 plasmid, or miR-15b-5p mimics+pcDNA3.1/ACSS2 plasmid were added into the plates. The transient transfections of these mimics or inhibitors were performed using the Exfect Transfection reagent (Vazyme Biotech Co.) according to the manufacturer's instructions.
4
Investigating the Effects of DZNeP and Ven on Apoptosis
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DZNeP (Cat.No S7120) and Ven (Cat.No S8048) were purchased from Selleck (Shanghai, China). For in vitro experiments, DZNeP and Ven were dissolved in anhydrous dimethyl sulfoxide (DMSO). IMDM was taken from HyClone Cytiva (Shanghai, China), and other mediums and FBS were obtained from Gibco (Beijing, China). The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). For flow cytometry antibodies, mouse anti-human MAbs and apoptosis antibodies were purchased from BD Biosciences (BD, USA), including anti-CD34-FITC, anti-CD117-PE, the FITC Annexin V apoptosis Detection Kit, APC Annexin V, and 7-AAD. The antibodies for the western blot were cleaved caspase-3 (9664, CST), PARP (9542, CST), BCL-2 (Ab32124, Abcam), BAX (Ab32503, Abcam), MCL1 (66026-1-Ig, Proteintech), PIK3IP1 (NBP1-69623, Novusbio), and GAPDH (Ab181602, Abcam). The PCR primers of EZH2, phosphoinositide-3-kinase-interacting protein1 (PIK3IP1), and GAPDH were synthesized by Sangon Biotech (Shanghai, China). The shRNA plasmid to PIK3IP1 was purchased from Corues Biotechnology (Nanjing, China). The ExFect transfection reagent was obtained from Vazyme (Nanjing, China).
5
Wound Healing Assay for Cell Migration
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Wound healing assays were used to assess the migration of SiHa and CaSki cell lines. Cells were transfected with sh-TXNDC12 plasmids using the ExFect Transfection Reagent (Vazyme, China) for 48 h for subsequent experiments. When the cell density in the 12-well plate grows to 80%–90%, we use the sterile tip of a pipette to draw a vertical line straight down the center of the 12 wells, taking care to keep the same width in each well. The area of the cell scratch was taken pictures to record when the line is drawn. In 24 h later, the cell scratch area was taken pictures again to record. Cell migration rate/Wound Healing = (0 h scratch width—24 h scratch width)/0 h scratch width * 100%. Scratch Wound Healing Assay was quantified using ImageJ (ImageJ software, United States).
6
SOCS5 Regulation of NDRV Infection
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RT-qPCR was performed with total RNA extracted from DEFs as the template to amplify the SOCS5 coding region. Subsequently, the PCR products were inserted into pcDNA3.1(+) vector to achieve eukaryotic expression in DEFs. Small interfering RNA (siRNA)-SOCS5 were provided by Shanghai GenePharma Co, Ltd. DEFs were transfected with pcDNA3.1(+) or pcDNA3.1(+)-SOCS5 with ExFect® Transfection Reagent (Vazyme, China). At 6 h after transfection with pcDNA3.1(+)-SOCS5 or siRNA-SOCS5, DEFs were treated with NDRV for the indicated time. Subsequently, RT-qPCR and western blot were performed to analyze the function of SOCS5 in virus propagation and IFN-I expression.
7
Targeted Knockdown of mRNA Using shRNA
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According to the mRNA sequences in GenBank, we designed 2 different small haircut RNA (shRNA) sequences by using the online design software of BLOCK-iT™ RNAi Designer (http://rnaidesigner.thermofisher.com/rnaiexpress/ (accessed on 9 February 2023)) for each mRNA sequence. The nucleotide sequences are shown in Supplementary Table S1 . Human glioma cells were plated at a density of 4 × 105 per 60 mm dish for 18 h before transfection. The transfection was performed using ExFect transfection reagent (Vazyme, Nanjing, China) according to the manufacturer’s instructions.
8
Investigating DLEU7-AS1 Regulation of miR-26a-5p and Coronin-3 in Cancer Cells
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The 786-O or Caki-1 cells (1 × 106/well) with shNC or sh DLEU7-AS1 were seeded in 96-well plates. miR-26a-5p inhibitor, coronin-3 expression plasmid or their control (4 μg) was transfected using Exfect Transfection Reagent (Vazyme, Nanjing, Jiangsu, China). After 48 h of transfection, the CCK8 array (Yeasen, Shanghai, China) was used to detect cell viability following the manufacturer's instructions. A SpectraMax absorbance reader (Molecular Devices, San Francisco, CA, USA) was used to measure absorbance at 450 nm. The cell cycle and apoptosis were measured using the NovoCyte flow cytometer (Cat#: 1300, ACEA, San Diego, CA, USA) with the Annexin V-FITC/PI apoptosis detection kit (Cat#: A211-02, Vazyme, Nanjing, China). The flow cytometry data were analyzed using NovoExpress 1.4.1 (ACEA).
9
Lentiviral shRNA Knockdown of SEC61A1
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Following the manufacturer’s instruction, we constructed lentiviral shRNA plasmids for SEC61A1 by subcloning the shRNA oligos into the lentiviral shRNA vector with an IRES GFP (pLV3ltr-ZsGreen-Puro-U6) (Corues Biotechnology, China). The shRNA oligos design for SEC61A1 is listed in Table S1.
Following the manufacturer’s instructions, 293T cells were transfected with the indicated viral plasmid and psPAX2 and pMD2.G (packaging plasmids) (Corues Biotechnology, China), via Exfect Transfection Reagent (Vazyme Biotech Co., Ltd, China, No. T101-01). After 48 and 72 h post-transfection, viral supernatant was collected and filtered with a 45 μM filter. AML cells were therefore transduced with the resulting lentivirus. HitransG P (KeyGen BioTECH, China) was added to increase the transduction efficiency. After 48 h transduction, 1 µg/mL of puromycin (InvivoGen, CAS.58-58-2) was added into the infected cells for stable expressing cell selection. Via flow cytometry, the efficiency of transduction was monitored by GFP (+) cells, more than 95% of GFP (+) cells were used for knockdown efficiency and further in vitro experiments.
Following the manufacturer’s instructions, 293T cells were transfected with the indicated viral plasmid and psPAX2 and pMD2.G (packaging plasmids) (Corues Biotechnology, China), via Exfect Transfection Reagent (Vazyme Biotech Co., Ltd, China, No. T101-01). After 48 and 72 h post-transfection, viral supernatant was collected and filtered with a 45 μM filter. AML cells were therefore transduced with the resulting lentivirus. HitransG P (KeyGen BioTECH, China) was added to increase the transduction efficiency. After 48 h transduction, 1 µg/mL of puromycin (InvivoGen, CAS.58-58-2) was added into the infected cells for stable expressing cell selection. Via flow cytometry, the efficiency of transduction was monitored by GFP (+) cells, more than 95% of GFP (+) cells were used for knockdown efficiency and further in vitro experiments.
10
Antibody Generation and Cell Lysis
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Rabbit polyclonal antibodies against VP1, VP2, VP3, and VP4 were all generated from rabbit serum by immunization with corresponding purified protein, respectively. Horseradish peroxidase (HRP) labeled anti-rabbit (IgG) was purchased from KPL (Millford, MA, USA). Cell lysis buffer NP-40 (50 mM Tris [pH 7.4], 150 mM NaCl, 1% NP-40) used for western blotting was purchased from Beyotime (P0013F, Shanghai, China). Immunofluorescence secondary antibody fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit antibody (A0562) was purchased from Beyotime (Shanghai, China). The Exfect Transfection Reagent (T101-01/02) was purchased from Vazyme Biotechnology (Nanjing, China).
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