The sequence of lncRNA PCAT-1 was amplified using PCR with a human genomic DNA from A549 cells as the template. One Step Cloning Kit ClonExpress II (Vazyme Biotech, Nanjing, P. R. China) was used. The corresponding reverse primer for the lncRNA PCAT-1 was 5′-CGGGTCGACTCTAGAGGTACCTAGGCTCAAACACACATTTATTCATC-3′, and the forward primer was 5′-CGAGAATTCACGCGTGGTACCACACATGGATATTGGATATCTGCATA-3′. The sequence of lncRNA PCAT-1 was then inserted into the KpnI site of the PCI mammalian expression plasmids (Thermo Fisher Scientific) and verified using sequencing. Two effective sequences of shRNAs (Sangon Biotech) for PCAT-1 were proven and inserted into a shRNA eukaryotic expressing vector. One sequence is 5′-TTGAGTACAAAGAGCTACCTATGGTTCAAGAGAGGTATCCATCGAGAAACATGAGTTTTTTTT-3′, and the other sequence is 5′- CTTCCCATGTGCCTCTAAGTGTTCAAGAGAGTGAAT-CTCCGTGTACCCTTCTTTTTT-3′. Additionally, the p-MIR-reporter plasmid (Promega, Madison, WI, USA) containing the 3′-UTR of RAP1A was constructed according to the above methods. The corresponding reverse primer for RAP1A 3’-UTR was 5′-TAGGTTTAAACAGTTAAGCTTACTGTTTAAAATGGGAAGGTTTATTAAG-3′, and the forward primer was 5′-AAAAGATCCTTTATTAAGCTTGTGAATCTCCGTGTACCCTTCG-3′. The binding-site mutant luciferase plasmid (binding site: GGAUGC replaced by CCTACG) was also transfected as a control.
P mir reporter plasmid
Manufactured by Promega
Sourced in United States
The P-MIR-reporter plasmid is a lab equipment product designed for gene expression studies. It contains a promoter and a reporter gene, enabling the monitoring of gene expression levels in cell-based experiments. The core function of this plasmid is to serve as a tool for researchers to investigate gene regulation and activity.
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Lab products found in correlation
6 protocols using p mir reporter plasmid
1
Cloning and Silencing of lncRNA PCAT-1
2
Validating circ_0005529 Binding Sites
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Amplified from a human genomic DNA, circ_0005529 sequence was inserted into the p-MIR-reporter plasmid (Promega). The binding sites: CUUUGCA was substituted by GAAACGU and used as a mutant. Similarly, the 3′-UTR p-MIR-reporter plasmids of different genes were obtained. Additionally, the binding sites: CUUUGCA of Sp1 luciferase plasmid was substituted by GAAACGU and used as a negative control. Table S2 showed the relevant primer sequences.
3
Luciferase reporter assay for HAS2 3'UTR
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The sequence of circ_102002 was amplified using a human genomic DNA and inserted into the p-MIR-reporter plasmid (Promega, Madison, WI, USA). The mutant luciferase plasmid (binding site: UGGCCUUUCA substituted by AUCGGAAAGU) was also used as a control. According to the methods above, the p-MIR-reporter plasmid containing the 3ʹ-UTR of HAS2 was obtained. In addition, the corresponding mutant luciferase plasmid (binding site: UCAAGGAAAAGUUCUUUCA substituted by UCAAGAAAAGUUGAAAGU) was constructed as a negative control. Primers sequences were listed in Table S3 .
4
Luciferase Reporter Assay for miR-137 Targets
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The Src 3’UTR gene fragment was artificially synthesized, and then introduced into pMIR-reporter plasmid (Promega, Madison, WI, USA) within the endonuclease site SpeI and Hind III. After restriction enzyme cutting, wild-type sequence and a mutant target fragment of Src were inserted into the pMIR-reporter reporter plasmid using T4 DNA ligase. Wild-type Src luciferase reporter plasmid and mutant luciferase reporter plasmids were co-transfected with miR-137 into HEK-293T cells (Shanghai Beinuo Biotechnology Co., Ltd., Shanghai, China) respectively for 48 h. Next, the cells were collected. Luciferase activity in cell extracts was analyzed by Dual-Luciferase Reporter Assay System (Promega Corp., Madison, Wisconsin, USA).
5
Dual-Luciferase Assay for miR-133a Binding
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The putative miR-133a target binding sequence in ABHD11-AS1 and its mutant of the binding sites was amplified by PCR and the PCR products were cloned into pMirReporter plasmid (Promega, Madison, USA) to form the ABHD11-AS1-wild-type (WT-ABHD11-AS1) vector and ABHD11-AS1-mutated-type (MUT-ABHD11-AS1). Similarly, the SOX4-wild-type (WT-SOX4) and SOX4-mutated-type (MUT-SOX4) reporter vectors were constructed. Subsequently, mutated or wild-type pMirReporter luciferase vector and miR-133a mimic or NC-mimic were cotransfected into HEK-293 cells in 96-well plates for 24 h with Lipofectamine 3000 (Invitrogen). The luciferase activity was determined by the luciferase reporter assay system (Promega) according to the manufacture’s instruction.
6
Validation of miR-1192 Targeting CXCR4
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The Bioinformatics website, microRNA.org was used to collect the analysis of the targeting gene of miR-1192, in order to predict the targeting relationship between both CXCR4 and miR-1192. A dualluciferase reporter assay was performed for further verification of the targeting relationship. CXCR4 3'UTR was synthesized, digested with SpeI and HindIII, and introduced into the pMIR-Reporter plasmid (Promega, Madison, WI, USA). Using the wild-type (WT) sequence and CXCR4 as the reference, we were able to design the mutated complementary sequence of the seed sequence. Following digestion by restriction endonucleases, the mutated fragment was then inserted into the pMIR-Reporter plasmid using T4 DNA ligase. Both the cell number and transfection efficiency were normalized using a pRL-TK (TaKaRa Biotechnology Ltd., Dalian, China), with Renilla luciferase being used as an internal reference. The vaginal epithelial cells in the MiR-1192 mimics and negative control groups were respectively co-transfected with the luciferase reporter vector. After transfection had been completed for a duration of 48 h, the cells were collected and lysed. Luciferase activities were determined by the Luciferase Reporter Assay System (Promega, Madison, WI, USA).
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