NC mimics, miR-107 mimics, miR-613 mimics, miR-206 mimics, miR-338-3p mimics, AKAP12-pcDNA3.1, sh-AKAP12-pcDNA3.1 and pcDNA3.1 plasmid vector were all obtained from GenePharma (Shanghai, China). Before transfection, cells were incubated in a 6-well plate in 5% CO2 at 37°C for 18–24 h. Lung adenocarcinoma cells were firstly transfected with pcDNA3.1 plasmid vector as the control, AKAP12-pcDNA3.1 as the AKAP12 group, and sh-AKAP12-pcDNA3.1 as the sh-AKAP12 group, respectively. Cancer cells were also transfected with 100 nM NC mimics, miR-107 mimics, miR-613 mimics, miR-206 mimics or miR-338-3p mimics respectively by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Lung adenocarcinoma cells comprised four groups: cells co-transfected with 50 nM NC mimics and 2 μg of pcDNA3.1 plasmid vector were the control group, cells co-transfected with 50 nM NC mimics and 2 μg of AKAP12-pcDNA3.1 were the AKAP12 group, cells co-transfected with 50 nM miR-338-3p mimics and 2 μg pcDNA3.1 plasmid vector were the miR-338-3p mimic group, and cells co-transfected with 2 μg of AKAP12-pcDNA3.1 and 50 nM miR-338-3p mimics were the AKAP12 + miR-338-3p mimic group.
Mir 338 3p mimic
Manufactured by GenePharma
Sourced in China
The MiR-338-3p mimic is a laboratory product that contains a synthetic RNA molecule designed to mimic the function of the endogenous miR-338-3p microRNA. MicroRNAs are small, non-coding RNA molecules that play a role in regulating gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Mir 338 3p inhibitor, by GenePharma (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Pcdna3, by GenePharma (2 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Mir 107 mimics, by GenePharma (1 mentions)
Mir 613 mimic, by GenePharma (1 mentions)
Mir 206 mimic, by GenePharma (1 mentions)
Lipofactamine 2000, by Thermo Fisher Scientific (1 mentions)
Sh nr2f1 as1, by GenePharma (1 mentions)
Sh ccnd1, by GenePharma (1 mentions)
Pcdna3.1 nr2f1 as1, by GenePharma (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Mir nc mimic, by GenePharma (1 mentions)
Anti nc, by GenePharma (1 mentions)
Si circ 3, by GenePharma (1 mentions)
Anti mir 338 3p, by GenePharma (1 mentions)
Si circ 1, by GenePharma (1 mentions)
Si circ 2, by GenePharma (1 mentions)
Si nc, by GenePharma (1 mentions)
12 protocols using mir 338 3p mimic
1
Modulation of AKAP12 and miR-338-3p in Lung Cancer
2
Silencing NR2F1-AS1 and CCND1 in Thyroid Cells
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ShRNA negative control (NC), sh‐NR2F1‐AS1, sh‐CCND1, miR‐338‐3p mimics, miR‐338‐3p inhibitor and NC were all provided by GenePharma Co, Ltd (Shanghai, China). In brief, sh‐NR2F1‐AS1 was synthesized and loaded into the pENTRTM/U6 vector. After finishing this construction, pENTRTM/U6‐sh‐NR2F1‐AS1 was sequenced to verify the accuracy of the inserted sequence. Thereafter, pENTRTM/U6‐sh‐NR2F1‐AS1 was introduced into FTC‐133 cells and B‐CPAP cells using Lipofactamine2000 (Invitrogen, Carlsbad, CA). Stably transfected cell with antibiotic resistance were sifted and enriched by adding puromycin to culture medium. Based on protocols of Lipofectamine 2000 (Invitrogen), miR‐338‐3p mimics, miR‐338‐3p inhibitor and NC were introduced into FTC‐133 cells and B‐CPAP cells. PcDNA3.1‐NR2F1‐AS1 and pcDNA3.1‐CCND1 were from GenePharma as well. Before plasmid transfection, FTC‐133 cells and B‐CPAP cells were suspended and seeded in six‐well culture plates at 37°C. When cell confluence rate reached 80% to 90%, cell culture media should be replaced by serum‐free fresh medium 3 hours before transfection.
3
Validation of CircRNA-miRNA-mRNA Interactions
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The targeted binding site between circ_0003747 and miR-338-3p was predicted through the online website CircInteractome (https://circinteractome.irp.nia.nih.gov/ ). Meanwhile, the StarBase online website (http://starbase.sysu.edu.cn/index.php ) was utilized for predicting the binding site between miR-338-3p and PLCD3. The wild-type and mutant-type full-length sequences involving the predicted binding sequence of circ_0003747 (circ_0003747-WT/circ_0003747-MUT) and PLCD3 3’-UTR (PLCD3-WT/PLCD3-MUT) were cloned into the Xhol I-Not I restriction sites of pmirGLO firefly luciferase plasmid. All constructs were sequenced to verify the integrity. Lipofectamine 3000 (Invitrogen, USA) was used for Circ_0003747-WT/circ_0003747-MUT and miR-338-3p mimics (GenePharma, China) co-transfection. Luciferase assay was then carried out using a dual-luciferase reporter assay kit according to the manufacturer’s protocol (Promega) 48 h after transfection [25 ]. Data were normalized to Renilla Iuciferase activity and the relative activities of Luciferase were calculated. Besides, the correlation between miR-338-3p and PLCD3 was validated using the same method.
4
Regulating circ_0038467 and miR-338-3p in 16HBE cells
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For circ_0038467 knockdown studies, 16HBE cells were transiently transfected with small interfering RNA (siRNA) against circ_0038467 (30 nM, si‐circ#1, si‐circ#2 and si‐circ#3, GenePharma, Shanghai, China), and a scrambled oligonucleotide sequence (30 nM, si‐NC, GenePharma) were used as the negative control. For miR‐338‐3p overexpression studies, 16HBE cells were introduced with commercial miR‐338‐3p mimic (30 nM, GenePharma) or a nontarget negative control (30 nM, miR‐NC mimic, GenePharma). MiR‐338‐3p knockdown cells were produced using a commercial inhibitor of miR‐338‐3p (30 nM, anti‐miR‐338‐3p, GenePharma), with a matched scrambled sequence (30 nM, anti‐NC, GenePharma) as the negative control. The HiPerFect transfection reagent (Qiagen, Surrey, UK) was used for each transfection, referring to the protocols of producers.
5
Silencing KIFC1 and Overexpressing miR-338-3p
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The small interference RNA (siRNA) sequences were designed for KIFC1: sense 5′-UCG AAA UGA GAA AUC UCG GAG-3′, antisense 5′-CCG AGA UUU CUC AUU UCG AAU-3′. The negative control siRNA (siRNA-NC) were random sequences that have no homology with any known mammalian gene. KIFC1 siRNA (si-KIFC1) and negative control (siRNA-NC) were synthesized by GenePharma Company (Shanghai, P.R. China). To overexpress miR-338-3p, miR-338-3p mimic and mimic negative control (miR-NC) were purchased from GenePharma. 786-O, 769-P, and OS-RC-2 cells were seeded at 1 × 105 cells per well in six-well plates and incubated overnight at 37°C with 5% CO2. Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used to transiently transfect cells with si-KIFC1, siRNA-NC, miR mimic, or miR-NC (GenePharma). The efficiency of transfection was evaluated by real-time quantitative PCRRT-qPCR and Western blot analysis after 48 h of transfection.
6
Circular RNA Regulation in Cancer
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circ_0001018 shRNA, circ_0001018 overexpression vectors, SOX4 overexpression vectors, miR-338-3p mimic, and miR-338-3p inhibitor were purchased from GenePharma (Shanghai, China). For the construction of the circ_0001018 overexpression and SOX4 overexpression vectors, the full length of circ_0001018 and SOX4 was amplified by PCR, and the primers containing BamHI and XhoI sites were designed. Next, the full length of circ_0001018 and the full-length of SOX4 were inserted into pLCDH-cir (GenePharma, China) and pcDNA3.1 (GenePharma, China), respectively, at the BamHI and EcoRI sites. Finally, puromycin was used to select the transfected cells. As for circ_0001018 shRNA, two shRNAs for knockdown of circ_0001018 were designed, synthesized, and inserted between the BamHI and EcoRI sites of the pLV-CMV-puro-U6-shRNA lentiviral vector. Puromycin also used to screen out the transfected cells. The sequences of circ_0001018 shRNA (sh-1 and sh-2), circRNA-OE, miR-338-3p mimic, and miR-338-3p inhibitor are given in Table S2 . The cell transfection was performed using circ_0001018 shRNA (sh-1 and sh-2), circ_0001018 overexpression vectors, SOX4 overexpression vectors, miR-338-3p mimic, and miR-338-3p inhibitor. After transfection for 48 h using Lipofectamine 3000 (Invitrogen), the transfected cells were collected to assess the transfection efficiency by qRT-PCR.
7
Overexpression and Knockdown Plasmid Construction and Transfection
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The circ-0007022-overexpression/knockdown plasmids and the corresponding vectors were successfully constructed using GENE (Shanghai, China), and the miR-338-3p mimic, siRNA-NRP1, and their corresponding negative controls were purchased from GenePharma (Shanghai, China). Kyse150 and TE1 cells (1.5 × 105 cells/well) were seeded into six-well plates and incubated for 8 h before transfection. 20 ul of 1 × 107 TU/ml of plasmids were infected into cells and cultured with 1 ml DMEM with 10% FBS and 6 ug/ml polybrene (GENE) for 48 h, followed by selection using 8 μg/ml puromycin (Thermo Fisher Scientific). 2.5 ul of 20 um miRNA mimics, miRNA labeled with biotin at the 3′ end or siRNAs were injected into cells with 7.5 ul of Lipofectamine 3000 Reagent (Invitrogen) and 500ul of opti-MEM (Gibco), and the transfection efficiency was determined by qRT-PCR analysis after 24 h of transfection. Sequences of mimic, inhibitor, and vectors were listed in Supplementary Table S2 .
8
Transfection of miR-338-3p Mimic and Inhibitor
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The miR-338-3p mimic, inhibitor, and corresponding negative control (NC) were purchased from GenePharma (Shanghai, China). 50 nM miR-338-3p/NC mimics or inhibitor was transfected into cells (2 × 105/well) seeded in a 6-well plate, using Lipofectamine 3000 reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer's protocol. Cells were incubated at 37°C with 5% CO2 and harvested at least 48 h later for the subsequent study.
9
Regulation of HIF1α by miR-338-3p
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miR-338-3p mimic, miR-338-3p inhibitor, miR-338-3p mutant and negative control (NC) were purchased from Shanghai Gene-Pharma Co. (Shanghai, China). HIF1α-siRNA (sc-35561) and control siRNA-A (sc-37007) were purchased from Santa Cruz Biotechnology, Inc. HIF1α plasmid (18949) [21] (link) and hypoxia response element (HRE)-luc pGL vector (26731) [22] (link) were purchased from Addgene (Cambridge, MA). Control pGL vector were obtain from Promega (Madison, WI). The 3′UTR of HIF1A was PCR-amplified from HepG2 cDNA and cloned downstream of the luciferase gene in the pGL vector (Promega). Plasmid, siRNA, and miRNA transfection was performed using Lipofectamine 2000 (Invitrogen). Cells were subjected to functional or mechanistic analyses two days post-transfection. Luciferase activity was measured using the dual-luciferase reporter system (Promega). Renilla activity was used to normalize the relative firefly luciferase values.
10
CircRNA-Mediated Regulation of MAPK1 in LPS-Induced Inflammation
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Small interfering RNA (siRNA) targeting circ_0001679 (si-circ_0001679#1, si-circ_0001679#2 and si-circ_0001679#3), circ_0001679 overexpression vector (circ_0001679), the scrambled siRNA negative control (si-NC), miR-338-3p mimic, miR-338-3p inhibitor and miRNA negative control (miR-NC), MAPK1 overexpression vector (MAPK1) and negative control were purchased from GenePharma (Shanghai, China). Cells were harvested by 0.25% trypsin digestion, and seeded into 6-well plate at the density of 3 × 105 cells/well. When cell confluence reached approximately 70%, the cells were transfected with different plasmids, siRNA or miRNA mimic/inhibitor using Lipofectamine 2000 reagent (Invitrogen, MA, USA) [25 (link)]. 48 hours post transfection, cells were treated with LPS (0.5 μg/ml) for 24 hours before functional assays. The sequences of synthesized oligonucleotides were displayed as below. Si-circ_0001679#1; 5’-AGACGAGAAUCCUGAGAAACA-3’, Si-circ_0001679#2 5’-CAAAGAGAGUUCGAAUAAAGG-3’; Si-circ_0001679#3 5’-GCAAAGAGAGUUCGAAUAAAG-3’; miR-338-3p 5ʹ-UCCAGCAUCAGUGAUUUUGUUG-3ʹ; miR-338-3p inhibitor 5ʹ-CAACAAAAUCACUGAUGCUGGA-3ʹ;circ_0001679 5ʹ-CTGAGCATCTTCCGTTTG-3ʹ; MAPK1 5ʹ-AGAACATCATTGGCATCA-3ʹ.
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