A 507-bp segment of the 3′-UTR of Apaf-1 that contained the presumed miRNAs binding sites was amplified by PCR using mouse cDNA as a template with the following using primer sets: forward, 5′-GGACTAGTCACACTAGACAGGCACTCCACCG-3′ reverse, 5′-CCCAAGCTTGTTCAGCAAGGAAAGGGCCACAA-3′ (the SpeI and HindIII restriction sites were added for cloning). The PCR products were inserted into the p-MIR-report plasmid (Ambion). To test the binding specificity, we mutated the complementary site of miR-23a/b from AATGTGA to TTACACT and the complementary site of miR-27a/b from TACTGTGA to ATGACACT. For luciferase reporter assays, HEK-293T cells were cultured in 24-well plates, and each well was transfected with 0.2 μg of firefly luciferase reporter plasmid, 0.2 μg of β-galactosidase (β-gal) expression vector (Ambion), and 20 pmol of a precursor miRNA oligo using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. The β-gal vector was used as a transfection control. At 24 h post transfection, the cells were assayed using luciferase assay kits (Promega, Madison, WI, USA). The data depicted are representative of five independent experiments performed on different days.
β galactosidase β gal expression vector
Manufactured by Thermo Fisher Scientific
Sourced in China
The β-galactosidase (β-gal) expression vector is a DNA construct used to produce the enzyme β-galactosidase in a host organism. β-galactosidase is a hydrolytic enzyme that cleaves the disaccharide lactose into glucose and galactose. The expression vector contains the necessary genetic elements, such as a promoter and a coding sequence, to facilitate the production of β-galactosidase in the host system.
Automatically generated - may contain errors
Lab products found in correlation
Luciferase assay kit, by Promega (4 mentions)
Pmir report plasmid, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Pgl3 basic reporter vector, by Promega (1 mentions)
Pre mir 19b, by GenePharma (1 mentions)
Anti mir 19b, by GenePharma (1 mentions)
Pmir reporter vector, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Pgl3 basic vector, by Promega (1 mentions)
5 protocols using β galactosidase β gal expression vector
1
Apaf-1 3'-UTR miRNA Binding Study
2
miRNA Regulation of HuR via Luciferase Assay
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Luciferase vectors used in this study were purchased from Genescript (China). Briefly, for miRNA binding site tests, pMIR-report luciferase vectors containing binding sites for miR-22 or miR-129 on HuR’s 3’-UTR were constructed. We also purchased mutant plasmid to test binding specificity. The miR-22 binding site was mutated from GGCAGCT to CCGTCGA, and the binding site of miR-129 was mutated from CAAAAA to GTTTTT. For the miR-22 promoter assay, miR-22 promoter regions containing different Jun binding sites were inserted into pGL3 basic reporter vectors (Promega, USA). When transfecting SW480 with luciferase vectors and small RNA oligos, we also co-transfected the cells with a β-galactosidase (β-gal) expression vector (Ambion) as a control. Luciferase activity was tested using a luciferase assay kit (Promega, USA).
3
Luciferase Reporter Assay for miR-19b Binding
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A luciferase reporter assay was performed as previously described39 (link) to test miR-19b binding to its target gene, human SOCS3. The entire human SOCS3 3’-UTR, containing a presumptive miR-19b complementary site (seed sequence, UUUGCAC), was PCR-amplified with the following primers:
sense: 5’-TGGGAGCTCAATGTCAGCCCAGTAAGTATTGGCCAGT-3’; antisense: 5’-GATAAGCTT-GTGCTCTTTATTATAAATTACTGAAATGTTTC-3’. Human genomic DNA was used as a template. PCR products were cloned into the pMIR-REPORT plasmid (Ambion), and insertion was confirmed by sequencing. To test binding specificity, the miR-19b seed sequence was mutated from UUUGCAC to AAACGTG. For luciferase reporter assays, 2 μg firefly luciferase reporter plasmid, 2 μg β-galactosidase (β-gal) expression vector (Ambion), and 100 pmol pre-miR-19b (GenePharma, Shanghai, China), anti-miR-19b (GenePharma), and scrambled ncRNA (GenePharma) were transfected into Caco2 cells or HT29 cells in 6-well plates using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. The β-gal vector was used as a control. Twenty-four hours after transfection, luciferase assays were performed (Promega, Madison, WI, USA). The reported data represent three independent experiments.
sense: 5’-TGGGAGCTCAATGTCAGCCCAGTAAGTATTGGCCAGT-3’; antisense: 5’-GATAAGCTT-GTGCTCTTTATTATAAATTACTGAAATGTTTC-3’. Human genomic DNA was used as a template. PCR products were cloned into the pMIR-REPORT plasmid (Ambion), and insertion was confirmed by sequencing. To test binding specificity, the miR-19b seed sequence was mutated from UUUGCAC to AAACGTG. For luciferase reporter assays, 2 μg firefly luciferase reporter plasmid, 2 μg β-galactosidase (β-gal) expression vector (Ambion), and 100 pmol pre-miR-19b (GenePharma, Shanghai, China), anti-miR-19b (GenePharma), and scrambled ncRNA (GenePharma) were transfected into Caco2 cells or HT29 cells in 6-well plates using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. The β-gal vector was used as a control. Twenty-four hours after transfection, luciferase assays were performed (Promega, Madison, WI, USA). The reported data represent three independent experiments.
4
Src 3'UTR Luciferase Assay
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The full‐length human Src 3′‐UTR and the mutant Src 3′‐UTR were cloned into the p‐MIR‐reporter vector (Ambion, Austin, TX), respectively. TE‐1 cells were co‐transfected with the p‐MIR‐reporter vector, β‐galactosidase (β‐gal) expression vector (Ambion), and 10 pmol of miR‐1 mimic or scrambled negative control RNA. Relative luminescences were collected 24 h posttransfection using the luciferase assay kit (Promega, WI).
5
Cloning and Characterization of miR-23b-27b Promoter
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Promoter of miR-23b-27b cluster was amplified by PCR from mouse genomic DNA. PCR primers were listed as follows: 5′-CGACGCGTCGGCAAACAGGCAGAAGCAC-3′ (forward) and 5′-CCGCTCGAGCGGCAACCACTCCCATCCACA-3′ (reverse). The PCR products were separated by agarose gel electrophoresis, and the DNA fragments then isolated and cloned into the restriction enzyme (MluI and XhoI) digested pGL3 Basic Vector (Promega, Madison, WI, USA). The construct was confirmed by sequencing. Site 1 was mutated from CACGTG to CTCGAG. HEK-293T cells were cultured in 24-well plates, and each well was transfected with luciferase reporter plasmid, β-galactosidase (β-gal) expression vector (Ambion), and c-Myc overexpression vector using the lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s instructions. The b-gal vector was used as a transfection control. At 24 h post transfection, the cells were assayed using luciferase assay kits (Promega). The data depicted are representative of three independent experiments performed on different days.
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