A human LC3B promoter fragment, demonstrated in previous work, was amplified from NB4 genomic DNA, verified by sequencing and cloned into pGL3-basic (Promega, Madison, WI, USA) to generate the construct. The 3'-UTR segment comprising 59 bp of the HOTAIRM1, E2F1, DRMA2 or ULK1 3'-UTR was synthesized and inserted into the psiCHECK2 control vector (Promega) for the miRNA functional analysis. The core sequence of HOTAIRM1 wild-type targeting miR-125b and miR-20a/106b was 5′-UGGAGUUGGGGGUUUCUGUAGGCACUUUA-3′. The targets point mutated vectors were mutated at five points of the 8 bp sequences complementary to the seed sections of miRNAs. The mutated core sequence of HOTAIRM1 targeting miR-125b and miR-20a/106b were 5′-GUUUGA GUUCC C-3′ and 5′-UUUCUGUAGCGUGA UUA-3′ respectively (bold bases were mutated). All the wild types and point mutations were confirmed by DNA sequencing. The lentivirus carrying mRFP-GFP-LC3 was purchased from Hanbio Co. Ltd (Hanbio, Shanghai, China).
Psicheck 2 control vector
Manufactured by Promega
Sourced in United States
The PsiCHECK-2 control vector is a dual-luciferase reporter vector used for monitoring gene expression in eukaryotic cells. It contains both the Renilla luciferase and firefly luciferase genes, allowing for the normalization of experimental results.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Pgl3 basic, by Promega (1 mentions)
Fugene hd, by Promega (1 mentions)
Prl sv40 vector, by Promega (1 mentions)
Dual luciferase reporter dlr assay system, by Promega (1 mentions)
Synergy 4 hybrid microplate reader, by Agilent Technologies (1 mentions)
Dual luciferase reporter assay, by Promega (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
7 protocols using psicheck 2 control vector
1
Cloning and Mutagenesis of miRNA Targets
2
Validating miRNA-Target Interactions
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The LHFPL3 3′-UTR sequence was predicted to interact with miR-218-5p and miR-138-5p, LHFPL3 with a mutated sequence containing the predicted target sites were synthesized and cloned into the XhoI and NotI sites of a psiCHECK-2 control vector (Promega, U.S.A.). These constructs were termed psiCHECK-2-LHFPL-3′UTR-WT and psiCHECK-2-LHFPL-3′UTR-Mut. In the reporter assay experiment, the U87 cells were plated onto 24-well plates and transfected with psiCHECK-2-LHFPL-3′UTR-WT or psiCHECK-2-LHFPL-3′UTR-Mut, and miR-218-5p mimics, miR-138-5p mimics or Negative mimics using FuGENEHD (Promega, U.S.A.). After transfection for 48 h, the cells were harvested and assayed on the Dual-Luciferase Reporter Assay system (Promega) according to the manufacturer’s instructions. Transfection was repeated in triplicate in three independent experiments.
3
Cloning PTEN 3'-UTR with miR-21 Site
4
Dual-Luciferase Reporter Assay for miR-182 Regulation
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Cells were cultured in 48-well plates until the cell growth reached approximately 80% confluence. The miR-182 (bta-miR-182, Bos taurus miR-182) mimic and CFL1-Luc or CFL1-del Luc were co-transfected into cells by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The transfection reagent was replaced with fresh GM 4~6 h after transfection. Next, the cells were washed with PBS and harvested using 200 mL passive lysis buffer (PLB) 24 h post-transfection and rocked for 30 min at room temperature. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter (DLR) Assay System (Promega, Madison, WI, USA). Both reporter activities were quantitated within the same sample of lysate prepared from cells co-transfected with psiCHECK-2 control vector and pRL-SV40 vector (Promega, Madison, WI, USA). The firefly luciferase activities were normalized to the Renilla luciferase activities in each well. Firefly luciferase luminescence was quenched by greater than 5 orders of magnitude.
5
Validating miR-200a Binding to β-catenin
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The β-catenin 3′-UTR sequences containing the predicted miR-200a binding sites were cloned into psiCHECK2 control vector (C8021, Promega, USA) to generate the plasmid psiCHECK2-WT-β-catenin 3′-UTR. The psiCHECK2-MUT-β-catenin 3′-UTR was generated from psiCHECK2-WT-β-catenin 3′-UTR by deleting the “ACTGTAC” binding site for miR-200a. For the luciferase reporter assay, HEK293 cells were coinfected with the luciferase reporter vectors or miR-200a mimics using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Luciferase activity was measured following infection for 48 h using the Dual-Luciferase Reporter Assay (Promega, Madison, WI, USA) with a luminometer (Synergy 4 Hybrid Microplate Reader, BioTek, Winooski, VT, USA). Luciferase activity values were normalized to Renilla values.
6
Validating miR-486-5p Targeting of KIAA1199
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We selected two potential miR‐486‐5p binding sites from online microRNA prediction databases to construct plasmids with KIAA1199 3′‐untranslated region (3′‐UTR) subcloned to the 3′‐end of a luciferase reporter downstream of the firefly luciferase gene. We synthesized a 1051‐bp sequence containing the projected miR‐486‐5p target sites and then clone it into the psi‐CHECK2‐control vector (Promega, Madison, WI, USA). Thus, we generated two psi‐CHECK‐KIAA1199‐3′‐UTR‐wild‐type and two psi‐CHECK2‐KIAA1199‐3′‐UTR‐mutant vectors. NSCLC cells were seeded onto 24‐well plates and co‐transfected with wild‐type or mutated plasmid and either miR‐NC or miR‐486‐5p mimics using Lipofectamine 2000 (Invitrogen). After incubation for 48 hours, the cell lysates were harvested. The firefly luciferase activity was assessed by the Dual‐Luciferase Reporter Assay Kit (Promega) and then standardized with renilla luciferase activity. Each experiment was performed thrice.
7
Luciferase Reporter Assay for let-7i Targeting
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Two oligonucleotides corresponding to a region containing a putative let-7i target site in the 3′-UTR of murine Myc mRNA were synthesized. The oligonucleotides were annealed and cloned into the XhoI and NotI sites located downstream of the luciferase open reading frame in the luciferase reporter psiCHECK-2 control vector (Promega, Madison, WI). When expressed in cells, this reporter construct produces a luciferase reporter mRNA harboring a Myc-derived putative let-7i targeting sequence in the 3′-UTR. This reporter (0.2 mg) was co-transfected into cells with different concentrations of let-7i mimic or inhibitor (usually 60 nM) together with a Renilla luciferase reporter (10 ng). Assays were performed 24 h after transfection using the dual luciferase reporter assay system (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity. The experiments were performed three times with three replicates each.
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