Luciferase reporter vectors LINC01123-WT/MT and TUFT1-WT/MT were constructed by inserting the wild-type (WT) LINC01123 or TUFT1 fragment and a LINC01123 or TUFT1 fragment containing mutated (MT) binding sites for miR-34a-5p into pmirGLO expression vectors (Promega, Madison, WI, USA). miR-34a-3p mimics or NC mimics were co-transfected with the above vectors for 48 h in 293T cells. The dual-Luciferase reporter assay system (Promega) was applied for detecting luciferase activity.
Pmirglo expression vector
Manufactured by Promega
Sourced in United States
The PmirGLO expression vector is a plasmid designed for the expression of genes in mammalian cells. It contains the monomeric Renilla luciferase (Rluc) gene as a reporter for monitoring gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (4 mentions)
Dual luciferase assay system, by Promega (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Pgl3 vector, by Promega (2 mentions)
Luciferase reporter system, by Promega (1 mentions)
Anti ago2, by Abcam (1 mentions)
Rip assay kit, by Merck Group (1 mentions)
Dual luciferase reporter system, by Promega (1 mentions)
Dharmafect transfection reagent, by Horizon Discovery (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Quickchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
12 protocols using pmirglo expression vector
1
Luciferase Reporter Assay for miR-34a Targets
2
Circular RNA and miRNA Binding Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The sequences of hsa_circ_0018189 and 3'untranslated region of xCT (xCT 3'UTR) containing the wild‐type (wt) miR‐656‐3p binding site were cloned into pmirGLO Expression vectors (Promega, Madison, WI), and the mutant type (mut) miR‐656‐3p binding site was performed based on the hsa_circ_0018189 wt and xCT 3'UTR wt vectors via QuikChange II Site‐Directed Mutagenesis Kit (Stratagene, Santa Clara, CA). Co‐transfection of wt/mut reporter vector and miR‐656‐3p mimic or mimic NC was performed. The transfected cells were collected and fully lysed, followed by low‐temperature and high‐speed centrifugation to collect the supernatant, which was then plated into the 96‐well plate in triplicate for the measurement of the luciferase activity using the luciferase reporter system (Promega).
RIP‐Assay Kit (Millipore) was used to perform RIP assay as per the manufacturer's instructions. After the beads were cleaned with cleaning buffer, pre‐diluted anti‐IgG (Abcam) or anti‐Ago2 (Abcam) solution was added to them and incubated at low temperature for 2 h. Cells were fully lysed after 48 h of transfection. The supernatant was incubated with the beads conjugated with antibodies at 4°C overnight. Then, the beads were collected by centrifugation at low temperatures and low speeds. RNA samples cleared from the beads were used for subsequent analysis of hsa_circ_0018189 and miR‐656‐3p.
RIP‐Assay Kit (Millipore) was used to perform RIP assay as per the manufacturer's instructions. After the beads were cleaned with cleaning buffer, pre‐diluted anti‐IgG (Abcam) or anti‐Ago2 (Abcam) solution was added to them and incubated at low temperature for 2 h. Cells were fully lysed after 48 h of transfection. The supernatant was incubated with the beads conjugated with antibodies at 4°C overnight. Then, the beads were collected by centrifugation at low temperatures and low speeds. RNA samples cleared from the beads were used for subsequent analysis of hsa_circ_0018189 and miR‐656‐3p.
3
Investigating CircRNA-miRNA Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Target miRNAs of circ_0097009 and target genes of miR-568 were forecasted by CircInteractome and Starbase. Wild sequences and mutant sequences of circ_0097009 or RNF38-3'UTR were inserted into pmirGLO expression vectors (Promega, Madison, WI, USA), namely WT-circ_0097009 and MUT-circ_0097009, WT-RNF38-3'UTR and MUT-RNF38-3'UTR. After that, the recombinant plasmid with miR-568 mimic or miRNA NC were cotransfected into HCC cells, and dual-luciferase reporter system (Promega) was applied to detect luciferase activity.
4
Validating miR-610 Binding to circ_0015756 and FGFR1 3'UTR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The sequence fragment of circ_0015756 containing miR-610 binding site and the corresponding mutant sequence fragment were synthesized and inserted into the pmirGLO expression vector (Promega, Madison, WI, USA), namely WT-circ_0015756 and MUT-circ_0015756. Similarly, WT-FGFR1 3ʹUTR and MUT-FGFR1 3ʹUTR were also generated following the same manner. Subsequently, SNU-387 and Huh7 cells were cotransfected with miR-610 mimic and WT-circ_0015756, MUT-circ_0015756, WT-FGFR1 3ʹUTR or MUT-FGFR1 3ʹUTR, and miRNA NC and these fusion plasmids were also cotransfected into SNU-387 and Huh7 cells as the control. After 48 h-transfection, the luciferase activity of cells was measured using the Dual-Luciferase Assay System (Promega).
5
Characterizing miR-140-5p Regulation of XIST and ORC1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PCR was conducted to amplify the fragment from XIST which contained the predicted miR-140-5p binding site, then the fragment was cloned into a pmirGLO Expression Vector (Promega, Madison, WI, USA) to form the XIST-wild-type reporter vector (XIST-wt). The miR-140-5p seed sequence binding site was mutated to create the corresponding mutant which was named as XIST-mut. 293T cells were co-transfected with miR-140-5p mimics or NC, and XIST-wt or XIST-mut plasmids using Lipofectamine 2000 (Invitrogen). For testing the relation between miR-140-5p and ORC1, the 3′-UTR region of ORC1 containing the binding site of miR-140-5p was constructed into the pGL3 vector (Promega). The cells were transfected with miR-140-5p mimics or NC, and ORC1-wt or ORC1-mut plasmids. The Dual-Luciferase Reporter Assay System (Promega) was used for conducting luciferase reporter assay.
6
Luciferase Reporter Assay for miR-1253 Targeting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The circ_0072995 or EIF4A3 sequence containing the binding element for miR-1253 was constructed into pmirGLO expression vector (Promega, Madison, WI, USA). For luciferase reporter assay, the luciferase vector and miR-1253 mimics were transfected into cells. After cultured for 48 h, the luciferase activity was measured using the Dual-Luciferase Assay System (Promega).
7
Validating miRNA-target interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In our recent study, we found that miR-141-3p and miR-429b-3p have higher expression level in XY than YY testis [23 (link)]. To investigate potential targets of miR-141-3p and miR-429b-3p, first the Open Reading Frame (ORF) and 3’UTR of YY highly expressed unigenes were predicted, by searching against the vertebrate genomic database in GENSCAN (http://genes.mit.edu/GENSCAN.html ). Perl scripts of both Target Scan and miRanda were performed for searching the putative targets with default parameters, including context score percentile ≥100 for Target Scanand Max_Energy≤ −20 and for miRandabased on hybrid energy and stability [44 (link),45 (link)]
To characterize the interaction between miR-141-3p/-429b-3p and their predicted target genes, the 3’-UTR of selected putative target genes (Itgb2 and Gria4a) were inserted into the pmirGLO expression vector (Promega, USA). Hek-293T cells were seeded in 96-well plates and co-transfected with the constructed vectors and microRNA mimics or its control oligonucleotide using DharmaFECT transfection reagent (Dharmacon). 36h post transfection, the dual-luciferase reporter assay system was used to detect reporter (Firefly and Renilla) activity as described [46 ]. The profile of relative luciferase activities were normalized to Renilla luciferase activities.
To characterize the interaction between miR-141-3p/-429b-3p and their predicted target genes, the 3’-UTR of selected putative target genes (Itgb2 and Gria4a) were inserted into the pmirGLO expression vector (Promega, USA). Hek-293T cells were seeded in 96-well plates and co-transfected with the constructed vectors and microRNA mimics or its control oligonucleotide using DharmaFECT transfection reagent (Dharmacon). 36h post transfection, the dual-luciferase reporter assay system was used to detect reporter (Firefly and Renilla) activity as described [46 ]. The profile of relative luciferase activities were normalized to Renilla luciferase activities.
8
miR-526b-3p Regulation of ADAM9
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The fragment from LINC00689 containing the predicted miR-526b-3p binding site was amplified by PCR and cloned into a pmir-GLO Expression Vector (Promega, Madison, WI, USA) to form the LINC00689-wild-type reporter vector (LINC00689-WT) containing a luciferase reporter gene. The corresponding mutant was created by mutating the miR-526b-3p seed sequence binding site, which was named LINC00689-MT. MGC-803 cells were cotransfected with the wild-type fragments and miR-526b-3p mimic using Lipofectamine 2000 (Invitrogen). To test the relationship between miR-526b-3p and ADAM9, the 3’-UTR of ADAM9 containing the binding site of miR-526b-3p was cloned into the pGL3 vector (Promega). The cells were transfected with miR-526b-3p mimics or mimics-ctrl and ADAM9-wild-type (ADAM9-WT) or ADAM9-mutant type (ADAM9-MT) plasmids. A luciferase reporter assay was performed using the Dual-Luciferase Reporter Assay System (Promega).
9
Luciferase Reporter Assay for LINC00885 and BAZ2A
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Full sequence of LINC00885 containing the predicted miR-3150b-3p binding sequences or muated miR-3150b-3p binding sequences were respectively cloned into the pmirGLO Expression Vector (Promega, Madison, WI, USA) to generate the LINC00885-WT/MUT reporter plasmids. Wide type BAZ2A 3’UTR with miR-3150b-3p binding site, or with miR-3150b-3p binding site 1/2 mutated was subcloned into pmirGLO vectors to construct BAZ2A-WT, BAZ2A-MUT-1 (with binding site 1 mutated), BAZ2A-MUT-2 (with binding site 2 mutated) and BAZ2A-MUT-1 + 2 (with binding sites 1 and 2 both mutated). These plasmids were co-transfected with miR-3150b-3p mimics or NC mimics respectively into HEK293T cells using the Lipofectamine 2000 (Invitrogen). A dual-Luciferase Reporter Assay System (Promega) was utilized to evaluate the luciferase activities, with Renilla luciferase activity as normalized control.
10
Validating Talin 1 3'-UTR Interaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The talin 1 3’-UTR fragment containing the seed sequence was amplified by PCR using cDNA from RWPE cells and the following primers: forward, 5′-CGAGCTCCAGTCCCGCAGTACAT-3′; reverse, 5′-GCCGCGGTGGGGGAAGATAGTAT-3′. The amplified fragment was cloned downstream of the luciferase-coding sequence in a pmir-GLO expression vector (Promega, Wisconsin, USA) at the sites of Sal I and Sac I endonucleases (Takara, Dalian, China). The vector containing the seed sequence was called pGL-TLN1. A control vector containing a mutated sequence generated by a quickChange™ Site-directed Mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA) was called pGL-mut. HEK293T cells were transfected with 100 ng pGL-TLN1 + NC RNA, pGL-TLN1 + miR-124, pGL-mut + miR-124, and pGL-mut + NC RNA. After 24 h, the cells were harvested and subjected to a Dual Luciferase Reporter Assay kit (Promega, Wisconsin, USA). The lysate was then analyzed by a bioluminescence detection system (Berthold Technologies, Bad Wildbad, Germany) to determine the relative light units.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!