Briefly, psiCHECK-2 reporters (Promega) with target sites for miR-9-alt or miR-9-can were inserted into the 3’UTR of the Renilla luciferase gene. Both strands of the target sequence were chemically synthesized, phosphorylated and ligated into the linearized psiCHECK-2 vector. Similarly, sh-miR-9-can/alt and sh-control were cloned downstream of a U6 promoter. Primers and oligonucleotides used in cloning are listed in Table S2 . 50 ng of the target reporter plasmids were co-transfected with either 50 ng of sh-miR-9-can/alt or sh-control into HEK293T cells. Cell lysates were obtained 48h post-transfection and measured with the Dual-Luciferase® Reporter Assay System (Promega).
Psicheck 2 reporter
Manufactured by Promega
Sourced in United States
The PsiCHECK-2 reporter is a dual-luciferase reporter vector used for gene expression analysis. It contains the Renilla luciferase gene as a reporter and the firefly luciferase gene as a control.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (3 mentions)
Dual luciferase detection kit, by Promega (1 mentions)
Lipofectamine 2000 reagent, by Promega (1 mentions)
Prl tk expressing renilla luciferase, by Promega (1 mentions)
Dreamtaq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Dual luciferase assay system, by Promega (1 mentions)
7 protocols using psicheck 2 reporter
1
Cloning and Luciferase Assay for miR-9 Targets
2
Cloning and Luciferase Assay for miR-9 Targets
3
Inhibition of miR-21 and ZIKV Infection
4
Evaluating lncRNA-miRNA Interaction Dynamics
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The target sequence was predicted by the bioinformatics website, and lncRNA SDCBP2-AS1 or EPDR1 3′-UTR sequence binding to miR-100-5p was amplified and mutated via site-directed mutagenesis kit (NBS, Beijing) to generate lncRNA SDCBP2-AS1 or EPDR1 3′-UTR mutant (MUT) sequence. Subsequently, the amplified lncRNA SDCBP2-AS1 or EPDR1 3′-UTR sequence and the lncRNA SDCBP2-AS1 or EPDR1 3̲′-UTR MUT sequence were inserted into the psi-CHECK2 reporter (Promega, WI, USA) and sequenced. Wild-type (Wt)-lncRNA SDCBP2-AS1 or Wt-EPDR1 3′-UTR and the Mut-lncRNA SDCBP2-AS1 or Mut-EPDR1 3′-UTR vectors were collected. Wt-lncRNA SDCBP2-AS1 or Wt-EPDR1 3′-UTR vector, or Mut-lncRNA SDCBP2-AS1 or Mut-EPDR1 3′-UTR vector, and miR-100-5p mimic or mimic-NC were co-transfected into cells via LipofectamineTM 2000. The samples were harvested 48 h after transfection. Finally, the relative luciferase activity of firefly and Renilla was detected with the dual luciferase detection kit (Promega).
5
Validating miR-520a-3p Regulation of EGFR
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TargetScan 7.2 (http://www.targetscan.org/vert_72/ ) was used to predict the potential targets of miR-520a-3p, and the binding sites between miR-520a-3p and EGFR were observed. To investigate the association between miR-520a-3p and EGFR, luciferase reporter plasmids was generated containing the 3′-UTR sequence of EGFR. Wild-type (WT) and mutant (MUT) 3′-untranslated regions (UTRs) of EGFR were amplified by PCR using DreamTaq DNA Polymerase (Thermo Fisher Scientific, Inc.) and then cloned into the psiCHECK-2 reporter (cat no. C8011; Promega Corporation, Madison, WI, USA). Y79 cells were co-transfected with mimic or NC and the mutant (MUT-EGFR) or wild-type (WT-EGFR) 3′-UTR of EGFR and 25 ng pRL-TK (expressing Renilla luciferase as the internal control; Promega Corporation) using Lipofectamine® 2000 reagent for 48 h. Luciferase activity was determined 48 h after cell transfection using the Dual-Luciferase Assay system (Promega Corporation,) on a luminometer (Mithras LB940; Berthold Technologies USA, LLC, Oak Ridge, TN, USA) according to the manufacturer's protocols and normalized to Renilla luciferase activity. Experiments were repeated in triplicate.
6
Inhibition of miR-21 and ZIKV Infection
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Hela cells were transfected with inhibitors targeting human miR-21 or non-targeting control A (Power inhibitors, Exiqon) at a final concentration of 25 nM using Lipofectamine RNAiMAX. 6 hours post transfection medium was replaced and cells were inoculated with ZIKV or re-transfected with ZIKV replicons as described above. miR-21 inhibition was validated using a psiCHECK-2 reporter (Promega) carrying a fully complementary miR-21 site at the 3' UTR of a Renilla luciferase reporter along with a Firefly reporter to normalise transfection efficiency. The miR-21 psiCHECK-2 reporter was deposited in addgene (plasmid number 114206). Luminescence was assessed using the Dual-reporter assay (Promega) and normalized to control psiCHECK-2 without the miR-21 binding site.
7
Dual-Luciferase Assay for ABCA1 and DANCR
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The 3′-UTR DNA fragment of wild-type or mutant ABCA1 was cloned into the psiCHECK2 reporter (Promega, WI, USA) as psiCHECK2-ABCA1-WT or psiCHECK2-ABCA1-Mut. The lnc-DANCR wild-type or mutant DNA fragment was also cloned into the psiCHECK2 reporter as psiCHECK2-DANCR-WT or psiCHECK2-DANCR-Mut.
HEK293 cells were co-transfected with miR-33a-5p mimic and psiCHECK2-ABCA1-WT, psiCHECK2-ABCA1-Mut, psiCHECK2-DANCR-WT or psiCHECK2-DANCR-Mut as well as pRT-TK Renilla plasmid. Luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega, WI, USA) after 48 h of co-transfection.
HEK293 cells were co-transfected with miR-33a-5p mimic and psiCHECK2-ABCA1-WT, psiCHECK2-ABCA1-Mut, psiCHECK2-DANCR-WT or psiCHECK2-DANCR-Mut as well as pRT-TK Renilla plasmid. Luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega, WI, USA) after 48 h of co-transfection.
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