Before the cell transfection, the astrocytes were grown in six-well plates and cultured to 80% fusion. The pmirGLO reporter vector containing a wild type (WT) or mutant type (Mut) miR-29a-5p binding sites in the 3′-UTR of LRP6, and named LRP6 WT or LRP6 Mut, was synthesized by Ribobio (Guangdong, China). miR-29a-5p mimic and the above recombinant vector were cotransfected into astrocytes using Lipofectamine 2000. Forty-eight hours after the transfection, the dual-luciferase reporter assay system (Thermo Fisher Scientific) was used to measure the relative luciferase activity.
Pmirglo reporter vector
Manufactured by RiboBio
Sourced in China
The PmirGLO reporter vector is a plasmid designed for luciferase reporter assays. It contains a multiple cloning site for inserting target sequences and a luciferase reporter gene to measure gene expression levels.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Mir 1 mimic, by RiboBio (2 mentions)
Pmirglo vector, by Promega (2 mentions)
Dual luciferase reporter assay system, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
4 protocols using pmirglo reporter vector
1
Luciferase Assay of miR-29a-5p Binding
2
Validating miR-411-3p Target Site
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Smurf2‐3′‐UTR wild‐type (Wt) plasmid or Smurf2‐3′‐UTR‐mutant (Mut) plasmid was synthesized and cloned into the pmirGLO reporter vector (Ribobio) containing the firefly luciferase gene and the control Renilla luciferase gene. Human embryonic kidney (HEK)293T cells were co‐transfected with the luciferase reporter vector and miR‐411‐3p negative control or miR‐411‐3p mimic using Lipofectamine 2000 reagent (11668‐019; Invitrogen, Thermo Fisher Scientific). At 48 h after transfection, luciferase activity was measured using the Dual‐Luciferase Reporter Assay System (E1910; Promega).
3
Dual-Luciferase Reporter Assay for miRNA Target Validation
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The 293T cell line used in this study was maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Waltham, United States) supplemented with 10% fetal bovine serum (Gibco), penicillin, and streptomycin in an incubator with 5% CO2 at 37°C. Predicted binding sites were cloned and inserted into the pmirGLO vector (Promega, Madison, United States). For reporter assays, 150 ng of pmirGLO reporter vector and 50 nM miR-1 mimic (RiboBio, Guangzhou, China) were cotransfected into 293T cells using Lipofectamine 2000. No-mimic-treatment cells were used as a blank control, and cells carrying the pmirGLO-Hsp vector alone were used as the negative control. Firefly and Renilla luciferase activities were measured 48 h post-transfection with a Dual-Luciferase Reporter Assay System (Promega). First, 100 μl of luciferase assay reagent II was added to each well; firefly luciferase activities were measured. Subsequently, 100 μl of Stop&Glo reagent was added, and Renilla luciferase activities were measured. Firefly luciferase in the pmirGLO vector was used for normalization of Renilla luciferase expression. Treatments were assessed in triplicate, and transfections were repeated three times. Firefly luciferase activities were divided by Renilla luciferase activities for each experiment, providing the ratio.
4
miR-1 Transcriptional Regulation Assay
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The 293T cell line used in this study was maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Waltham, USA) supplemented with 10% fetal bovine serum (Gibco), penicillin and streptomycin in an incubator with 5% CO 2 at 37°C. Predicted binding sites were cloned and inserted into the pmirGLO vector (Promega, Madison, USA). For reporter assays, 150 ng of pmirGLO reporter vector and 50 nM miR-1 mimic (RiboBio, Guangzhou, China) were cotransfected into 293T cells using Lipofectamine 2000. No-mimic-treatment cells were used as a blank control, and cells carrying the pmirGLO-Hsp vector alone were used as the negative control. Firefly and Renilla luciferase activities were measured 48 h posttransfection by Dual-Luciferase Reporter Assay System (Promega). First, 100 µL of luciferase assay reagent II was added to each well, firefly luciferase activities were measured, and 100 µL Stop&Glo reagent was then added. Renilla luciferase activities were then measured. Firefly luciferase in the pmirGLO vector was used for normalization of Renilla luciferase expression. Treatments were assessed in triplicate, and transfections were repeated three times. Firefly luciferase activities were divided by Renilla luciferase activities for each experiment, providing the ratio.
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