The potential target sites for miR-449a on the mouse SCN2B mRNA 3′-UTR seed regions, including both wild-type (wt) and mutant (mut) sequences, were cloned. The artificially cloned sequences were inserted downstream of the luciferase reporter gene, pGL3 (Guangzhou RiboBio Co., Ltd.), to generate the SCN2B 3′-UTR-wt and SCN2B 3′-UTR-mut vectors, as described previously (40 (link),41 (link)). Briefly, 293T cells (purchased from Institute of Biochemistry and Cell Biology, Shanghai, China) were seeded in 96-well plates and co-transfected with 100 ng/ml of each pGL3-SCN2B 3′-UTR-wt or -mut vector and 35 nM miR-449a mimics or NC (Guangzhou RiboBio Co., Ltd.). Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used as the transfection reagent. The effects of miR-449a treatment on luciferase activity were measured at 48 h post-transfection. The firefly and Renilla luciferase activities were separately measured using a Dual Luciferase Reporter Assay System kit (Promega Corporation) and a Tecan M200 luminescence reader (Tecan Group, Ltd.) according to the manufacturer's instructions. The relative transcriptional activity was normal-ized to Renilla luciferase activity.
M200 luminescence reader
Manufactured by Tecan
Sourced in Switzerland, United States
The Tecan M200 is a luminescence reader designed for sensitive detection of luminescent signals. It provides high-performance luminescence measurements for a wide range of applications in life science research and drug discovery.
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Lab products found in correlation
4 protocols using m200 luminescence reader
1
Characterization of miR-449a Binding Sites
2
Measuring miR-1 Regulation of G6PD
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Wild-type (wt) and mutant (mut) human G6PD mRNA 3′-UTR seed regions, which included the potential target site for miR-1, were cloned. The cloned sequences were inserted downstream of the pGL3 luciferase reporter gene to generate the G6PD 3′-UTR-wt and G6PD 3′-UTR-mut vectors. Briefly, 293T cells were seeded in 96-well plates and co-transfected with 100 ng/mL of the individual pGL3-G6PD 3′-UTR-wt/mut vectors and 50 nM miR-1 mimics or NC (Ribobio, Guangzhou, China). Forty-eight hours after transfection, the effects of miR-1 treatment on luciferase activity were measured using a dual-luciferase reporter assay system kit (Promega) and a Tecan M200 luminescence reader (Tecan Group Ltd, Männedorf, Switzerland) according to the manufacturer's instructions. Values were double-normalized to firefly luciferase activity and to cells transfected with empty control vectors [29 (link), 30 ].
3
PCGEM1 miRNA Regulation Analysis
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The whole mRNA sequences of the PCGEM1 gene were obtained by PCR amplification and cloned separately into multiple cloning sites of the psi-CHECKTM-2 luciferase miRNA expression reporter vector. HEK293T cells were transfected with miR-145 mimic, miR-145 inhibitor, a control miRNA, a miRNA inhibitor control, or empty plasmid using Lipofectamine 2000 according to the manufacturer’s instructions. Nucleotide-substitution mutation analysis was carried out using direct oligomer synthesis of PCGEM1 sequences. All constructs were verified by sequencing. Luciferase activity was measured using the dual luciferase reporter assay system kit (Promega Co, Madison, WI, USA) according to the manufacturer’s instructions on a Tecan M200 luminescence reader.
4
miRNA-Luciferase Reporter Assay
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The full sequence or 3′-untranslated region (3′UTR) of the gene was obtained by PCR amplification and cloned separately into the multiple cloning site of the psi-CHECKTM−2 luciferase miRNA expression reporter vector. 293T cells were transfected with an miRNA mimic, miRNA inhibitor, control miRNA, control miRNA inhibitor, or empty plasmid (50 nM; RiboBio, Guangzhou, China) using Lipofectamine 2000 according to the manufacturer's instructions. Nucleotide-substitution mutation analysis was carried out using direct oligomer synthesis of the full sequences or 3′UTR. All constructs were verified by sequencing. Luciferase activity was measured using a Dual Luciferase Reporter Assay System Kit (Promega, Madison, WI) on a Tecan M200 luminescence reader according to the manufacturer's instructions (7 (link)).
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