The wild type 3'UTR sequence of Bcl-w (wt 3 'UTR), which contains the putative miR-148b binding site, was amplified by PCR using the Bcl-w wt primer pair (Table 2 ). A mutated 3' UTR (mut 3' UTR) of Bcl-w was generated through site-directed mutagenesis with Bcl-w mut primer pair (Table 2 ) using a Quik-Change Site-Directed Mutagenesis Kit (Stratagene, USA). Both Bcl-w wt 3' UTR and Bcl-w mut 3' UTR were fused with the luciferase reporter gene in the psiCHECK-2 vector (Promega). Raji cells and SU-DHL-10 cells were divided into four groups. One group was co-transfected with Wt 3'UTR vectors, control vectors of psiCHECK-2 (Promega, USA) encoding Renilla luciferase and miR-148b mimic; one group was co-transfected with Wt 3'UTR vectors, control vectors of psiCHECK-2 encoding Renilla luciferase and miR-control; one group was co-transfected with mut 3'UTR vectors, control vectors of psiCHECK-2, and miR-control; and the fourth group was co-transfected with mut 3'UTR vectors, and a control vector encoding Renilla luciferase, control vectors of psiCHECK-2 (Promega, USA) and miR-control, with Lipofectamine 2000 (Invitrogen). After 48h, levels of luciferase activity were detected using the Dual-Luciferase Reporter Assay System (Promega) and normalized with the Renilla values. Values are presented as the ratio of firefly/Renilla values.
Psicheck 2
Manufactured by Promega
Sourced in United States, China, Japan, Germany
PsiCHECK-2 is a dual-luciferase reporter vector system designed for monitoring and analyzing gene expression and regulatory elements in cells. It contains a Firefly luciferase gene as a reporter and a Renilla luciferase gene as a control for normalization of transfection efficiency and cell viability.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (146 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (102 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (43 mentions)
Dual luciferase assay kit, by Promega (26 mentions)
Dual glo luciferase assay system, by Promega (26 mentions)
Psicheck 2, by Thermo Fisher Scientific (26 mentions)
Dual luciferase reporter assay kit, by Promega (25 mentions)
Dual luciferase reporter assay, by Promega (20 mentions)
Dual luciferase assay system, by Promega (16 mentions)
Dual luciferase reporter system, by Promega (12 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (11 mentions)
Psicheck 2 vector, by Promega (9 mentions)
Dual glo luciferase assay, by Promega (8 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (6 mentions)
Glomax 96 microplate luminometer, by Promega (6 mentions)
Microplate reader, by Molecular Devices (5 mentions)
Glomax, by Promega (5 mentions)
Pgl3 basic, by Promega (5 mentions)
Dual luciferase assay, by Promega (5 mentions)
Passive lysis buffer (plb), by Promega (5 mentions)
545 protocols using psicheck 2
1
Bcl-w 3'UTR Luciferase Assay Protocol
2
Validation of miR-4433 Binding to ABL1 3'UTR
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miR-4433 putative binding sites of ABL1 (3'UTR) was amplified by PCR from complementary DNA (cDNA) of K562 cells using the PrimeSTAR Max DNA Polymerase (Takara, Beijing). Mutant miR-4433 putative binding sequence of ABL1 (3'UTR) was generated by overlap-extension PCR method. The two sequences were inserted downstream of the renilla luciferase coding region between Xho I and Not I restriction enzyme sites of the psiCHECK2 (Promega, Beijing), and the constructed plasmids were nominated as psiCHECK2-ABL1-UTR and psiCHECK2-ABL1-UTRm. The transfection were carried out with the psiCHECK2, psiCHECK2-ABL1-UTR and psiCHECK2-ABL1-UTRm after 293T cells reached 50% confluence, and 6 hours later the miR-4433 and NC duplexes were transfected. Firefly and renilla luciferase activities were determined forty eight hours post transfection using Dual-Luciferase Reporter Assay System (Promega, Beijing) on a microplate reader (FLX800, BioTech, US). The relative luciferase activity was calculated by normalizing the renilla luciferase activity to firefly luciferase activity. See the primer sequences for PCR in Supplementary Table 1 .
3
Investigating TP63 Regulation of KRT10 Promoter
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KRT10 promoter, harbouring the predicted TP63 binding site, was cloned to psiCheck‐2 (Promega) to construct KRT10 promoter luciferase reporter vector (psiCheck‐2‐proKRT10). Cells were co‐transfected with pcDNA3.1‐TP63 or blank pcDNA3.1 and psiCheck‐2‐proKRT10 luciferase reporter vector; after 24 hours, the activities of firefly luciferase and Renilla luciferase were measured in the cell lysates using a Dual‐Luciferase Assay System (Promega).
4
Validating LINC01125-miR-1972 Interaction
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The interaction between LINC01125 and miR-1972 was verified by dual-luciferase reporter assays. LINC01125 containing wild-type or mutant miR-1972 binding sites was amplified and inserted into the luciferase vector psi-CHECK2 (Promega, Madison, USA) to generate the recombinant luciferase plasmids psiCHECK2-LINC01125-WT (LINC-WT) and psiCHECK2-LINC01125-WT (LINC-Mut). SKOV-3 and A2780 cells were seeded into 96-well plates at a density of 1×104 cells/well, maintained at 37°C for 24 h, and then transfected with LINC-WT or LINC-Mut and miR-1972 or miR-NC. Subsequently, the luciferase activities of Firefly and Renilla in treated SKOV-3 and A2780 cells were detected by the Dual-Luciferase Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity.
5
Deciphering miR-10b Binding Sites
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PsiCHECK2 miR-10b reporter with a perfect miR-10b-binding site has been produced as previously reported.7 (link) The IGFBP2 (NM_000597.3) open reading frame (ORF) was cloned into PsiCHECK2 (Promega) downstream of Renilla luciferase using the SgfI and Pmel restriction enzymes sites from pCMV6-XL4-IGBMP2 (#SC119778, Origine). Other fragments of IGFBP2 mRNA (including the ORF and the UTRs) and the PGK1 3′ UTR have been amplified with primers listed in Table S2 and cloned using the TOTO PCR kit (Invotrogen), cut with SgfI and Pmel restriction enzymes, and cloned into PsiCHECK2 (Promega) downstream of Renilla luciferase. The following fragments containing miR-10b putative binding sites have been produced: PGK1 (NM_000291.4) 2,421–3,190 (3′ UTR start) and 4,115–4,430 nt (3′ UTR end); IGFBP2 (NM_000597.3) 181–206 (V1) and 916–1,396 nt (common ORF + 3′ UTR); IGFBP2 (NM_001313990.2) 1–180 nt (V2); and IGFBP2 (NM_001313992.2) 1–423 nt (V3).
For the reporter assays, MCF7 cells were seeded at 5,000 cells/well in 96-well plates and co-transfected with 100 ng psiCHECK-2 luciferase reporters and either 50 nM miR-10b mimic or scramble mimic control (Ambion, Dharmacon). Luciferase luminescence was measured 24 h after transfection using Dual-Glo Luciferase Assay (Promega E2920) and GloMax explorer instrument (Promega). Renilla luciferase activity was normalized to the Firefly luciferase activity.
For the reporter assays, MCF7 cells were seeded at 5,000 cells/well in 96-well plates and co-transfected with 100 ng psiCHECK-2 luciferase reporters and either 50 nM miR-10b mimic or scramble mimic control (Ambion, Dharmacon). Luciferase luminescence was measured 24 h after transfection using Dual-Glo Luciferase Assay (Promega E2920) and GloMax explorer instrument (Promega). Renilla luciferase activity was normalized to the Firefly luciferase activity.
6
Investigating lncRNA DGCR5 Regulation by miRNAs and NF-κB1
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The fragment of lncRNA DGCR5 was amplified by PCR, cloned downstream of the Renilla gene in the psiCHECK-2 vector (Promega, Madison, WI, USA), and the resulting construct was named wt-DGCR5. To generate the DGCR5 mutant reporter, we mutated the seed region of DGCR5 to remove all complementarity to miR-21-3p and miR-23a-5p, and the resulting construct was named mut-DGCR5. These vectors were cotransfected into 293T cells with miR-21-3p/miR-23a-5p or anti-miR-21-3p/anti-miR-23a-5p. Luciferase assays were performed 48 h after transfection using the Dual-Luciferase Reporter Assay System (Promega). Renilla luciferase activity was normalized to firefly luciferase activity for each transfected well.
To investigate the binding of NF-κB1 to the DGCR5 promoter harboring the predicted NF-κB1 binding site, we cloned the DGCR5 promoter into psiCHECK-2 (Promega) to construct a DGCR5 promoter-containing luciferase reporter vector (psiCHECK-2-proDGCR5). Cells were cotransfected with these vectors and pcDNA3.1-NF-κB1 or blank pcDNA3.1. Luciferase assays were performed 48 h after transfection to determine the activities of firefly luciferase and Renilla luciferase.
To investigate the binding of NF-κB1 to the DGCR5 promoter harboring the predicted NF-κB1 binding site, we cloned the DGCR5 promoter into psiCHECK-2 (Promega) to construct a DGCR5 promoter-containing luciferase reporter vector (psiCHECK-2-proDGCR5). Cells were cotransfected with these vectors and pcDNA3.1-NF-κB1 or blank pcDNA3.1. Luciferase assays were performed 48 h after transfection to determine the activities of firefly luciferase and Renilla luciferase.
7
Dual-Luciferase Assay for miR-33-5p Binding
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The wild-type and mutant 3’UTR DNA fragments of JNK1 covering the predicted binding sites of miR-33-5p were successfully replicated. The psiCHECK™-2-JNK1-3’UTR (wild type and mutant) vector was constructed by combining the luciferase vector psi-CHECK™-2 (Promega, Madison, WI, USA) with JNK1-3’UTR (wild type and mutant). Similarly, we cloned the CDS of JNK1 or Lnc90386 into the pcDNA3.1 vector. The primers are described in Table 1
Dual-luciferase reporter assay is detailed in our previous reports (16 (link)). In a nutshell, when cells reached 80-90% confluence, using Lipofectamine 2000 (Invitrogen Life Technologies, USA) each co-transfected cells with wild-type or mutant reporter plasmid (200 ng) and 10 pmol of the indicated RNA oligonucleotides. Then, the luciferase activity in each group was detected by using an automatic microplate reader (Bio-Rad, Hercules, CA, USA) in accordance with the dual luciferase reporter gene detection kit instructions (Promega, Madison, WI, USA) according to the manufacturer’s protocol.
Dual-luciferase reporter assay is detailed in our previous reports (16 (link)). In a nutshell, when cells reached 80-90% confluence, using Lipofectamine 2000 (Invitrogen Life Technologies, USA) each co-transfected cells with wild-type or mutant reporter plasmid (200 ng) and 10 pmol of the indicated RNA oligonucleotides. Then, the luciferase activity in each group was detected by using an automatic microplate reader (Bio-Rad, Hercules, CA, USA) in accordance with the dual luciferase reporter gene detection kit instructions (Promega, Madison, WI, USA) according to the manufacturer’s protocol.
8
Dual-Luciferase Assay for miRNA-Target Interaction
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The WT-circ_0068087 and WT-ROBO1-3'UTR reporter plasmids were constructed through inserting the partial fragment of circ_0068087 or ROBO1 3'UTR into the psiCHECK2 (Promega, Madison, WI, USA) vector. The GeneArtTM Site-Directed Mutagenesis System (Invitrogen) was utilized to amplify the mutant fragment. The mutant fragment of circ_0068087 or ROBO1 3'UTR, including the mutant binding sites with miR-186-5p, was also inserted into the psiCHECK2 vector (Promega) to generate MUT-circ_0068087 and MUT-ROBO1-3'UTR. HUVECs were cotransfected with luciferase plasmids and miR-186-5p mimic or miRNA NC. The luciferase intensity was determined using a commercial Dual-Luciferase Reporter Assay Kit (Promega).
9
Cloning and Mutagenesis of 3' UTR Regions
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The 3′ UTR region of GATA3 or IL-4, as well as the 3′ UTR plus partial sequences in the coding region of IL-4, were cloned into psiCheck2 (Promega). miR-23a, miR-24, and miR-27a sequences were respectively cloned into pMDH-PGK-EGFP, similar to what was described previously (Lu et al., 2009 (link)). To generate GATA3 or IL-4 3′ UTR mutants, site-directed mutagenesis was performed (Agilent). 1 d before transfection, HEK293T cells were plated at 6.5 × 104 cells per well on a 24-well plate. psiCheck2 bearing WT 3′ UTR or corresponding mutant 3′ UTR were transfected to HEK293T cells with control vector or miRNA expressing plasmid using Fugene 6 (Promega). Luciferase activities were assessed at 24 h after transfection using Dual-Luciferase Reporter assay system (Promega) according to the manufacturer’s protocol.
10
Validating miR-322 Binding Sites in IGF1R and INSR 3'-UTRs
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Fragments of IGF1 receptor (IGF1R) 3′-UTR and insulin receptor (INSR) 3′-UTR containing putative miR-322 binding sites or mutational miR-322 binding sites were synthesized by Genewiz (Suzhou, China) and cloned into the psiCHECK2 vector (Promega, Madison, WI, USA) between the XhoI and NotI sites, respectively. These fragments are listed in Table 1 . All constructs were verified by DNA sequencing by Genewiz (Suzhou, China). HEK293T was transfected with 200 ng psiCHECK2-IGF1R-WT or psiCHECK2-IGF1R-Mut and 50 nM miR-322 mimic or mimic NC by using Lipofectamine 3000 Reagent in 96-well plates for 48 h. The activation of firefly and Renilla luciferase was analyzed by a dual-luciferase reporter assay kit (Promega) according to the manufacturer’s instructions.
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