To explore miR-8516 whether target gene STC2 and circRNA8220, 260 bp sequence of STC2 3′UTR and 344 bp total sequence of circRNA8220 were cloned into the psiCHECK-2 vectors (Addgene, CA, USA), respectively. The mutated plasmids with mutated target site (psiCHECK-2-Mut) were also constructed. The wild-type (psiCHECK-2-WT) or mutated (psiCHECK-2-Mut) plasmids were co-transfected with miR-8516 mimic or inhibitor into 293T cells using Lipofectamine™ RNAiMAX Reagent. At 24 h later, the result was obtained from thermo scientific varioskan flash (Thermo scientific, USA) using the Dual-Glo luciferase system (Promega, USA). The primer sequences are shown in Appendix A Table A1 . Each experiment was performed three times in triplicate.
Psicheck 2 vector
Manufactured by Addgene
Sourced in United States
The PsiCHECK-2 vector is a dual-luciferase reporter construct used for evaluating the activity of regulatory elements, such as promoters and enhancers, in eukaryotic cells. The vector contains both a firefly luciferase gene and a Renilla luciferase gene, allowing for the measurement of two distinct reporter activities within the same sample.
Automatically generated - may contain errors
Lab products found in correlation
Dual glo luciferase assay system, by Promega (2 mentions)
Fast mutation kit, by New England Biolabs (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Thermo scientific varioskan flash, by Thermo Fisher Scientific (1 mentions)
Dual glo luciferase system, by Promega (1 mentions)
Psicheck 2, by Thermo Fisher Scientific (1 mentions)
Synergy microplate reader, by Agilent Technologies (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Reporter lysis buffer, by Promega (1 mentions)
Bright glotm luciferase assay system, by Promega (1 mentions)
Glo lysis buffer, by Promega (1 mentions)
5 protocols using psicheck 2 vector
1
miR-8516 Regulation of STC2 and circRNA8220
2
Luciferase Assay for miRNA-574-5p Targets
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Before the luciferase assay, the psiCHECK-2 vectors (Addgene, Watertown, USA) of the EVI5L and circRNA-006258 were built. The miRNA-574-5p target sites were found from the former studies [11 (link)]. Primers of wild psiCHECK-2 vectors were designed and synthesized in the 3’ UTR of EVI5L and circRNA-006258 with special restriction enzyme sites: Xho I and Not I. The primers were used for psiCHECK-2 vectors in Table S1 . Then, when the GMECs had a density of 50,000 cells/well in 48-well plates, 0.33 mg psiCHECK-2-EVI5L and psiCHECK-2-circRNA-006258 were cotransfected, respectively, with 10 pmol miRNA-574-5p mimics or inhibitors into cells. After 24 h, renilla and firefly luciferase activities were measured using thermo scientific varioskan flash (Thermo scientific, Waltham, MA, USA) by the Dual-Glo luciferase assay system (Promega, Madison, WI, USA).
3
Functional Validation of miRNA Binding Sites
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Wild-type LINC01296 or MYCN 3′UTR sequences were amplified from a human cDNA library. Mutations of the miR-5095 binding site were introduced by site-directed mutagenesis using a fast mutation kit (New England BioLabs, Inc., Ipswich, MA, USA). The PCR fragment was cloned into psiCHECK-2 vector (Addgene, Inc., Cambridge, MA, USA) downstream of the firefly luciferase coding region within XhoI and NotI (Takara Bio, Inc., Otsu, Japan). psiCHECK-2-control was used as internal control. The psiCHECK-2 reporter plasmids (1 µg per well) were tranfected into 1×106 CCA cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). At 48 h after transfection, the luciferase activity was measured as previously described (25 (link)). The primer sequences for LINC01296 or MYCN cloning were as follows: LINC01296 (forward, 5′-CCCTGCAGCAGCAGCGGCGTG-3′ and reverse, 5′-ACACTCACACACACTCCCACC-3′) and MYCN (forward, 5′-ACGCTTCTCAAAACTGGACAG-3′ and reverse, 5′-AGCTATTTATTTTCATAAACATG-3′).
4
Regulation of ITGA6 3'UTR Activity
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The 3′ UTR of the ITGA6 gene was cloned from human umbilical vein endothelial cells into the psiCHECK-2 vector (Addgene_35672) to create psiCHECK-2-ITGA6-3′-UTR. 293 T and SV-HUC-1 cells were cultured, as described [18 (link)], and passaged at 80 % confluency. A total of 300,000 cells were plated in each well of a 24-well tissue culture plate. After 24 h, the cells were co-transfected with dCasRx-METTL3-CD, dCasRx-dMETTL3-CD, gRNAs, or psiCHECK-2-ITGA6-3′-UTR plasmids at a mass ratio of 5:3:1 using Lipofectamine 3000 (Life Technologies). After 48 h, the cells were washed with PBS and lysed with a reporter lysis buffer (Promega). Relative luciferase activity of the lysates was measured using the Dual-Glo luciferase assay system (Promega) on a SYNERGY microplate reader (BioTek, Winooski, VT, USA).
5
Luciferase Assay for miR-501-3p Binding
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Wild-type LINC00452 or ROCK1 3’UTR PCR fragments were cloned into psiCHECK-2 vector (Addgene, Inc., Cambridge, MA, USA) downstream of the firefly luciferase coding region within restriction sites XhoI and NotI (Takara Bio, Inc., Otsu, Japan). Corresponding mutations were introduced into the miR-501-3p binding site by site-directed mutagenesis using a fast mutation kit (New England BioLabs, Inc., Ipswich, MA, USA). The reporter plasmids were co-transfected with miR-NC, miR-501-3p mimics or miR-501-3p inhibitors, respectively, by LipofectamineTM 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were lysed 24 h using Glo Lysis Buffer (E266A, Promega) after transfection, and the luciferase activity of each extract was assayed using Bright-GloTM Luciferase Assay System (E2620, Promega).
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