The miR‐181a‐5p mimic (M) (miR10000256‐1‐5), miR‐181a‐5p inhibitor (I) (miR20000256‐1‐5), and corresponding control (MC, IC) (miR1N0000002‐1‐5, miR2N0000002‐1‐5) were obtained from Ribobio. YY1 overexpression plasmid was constructed from pEX‐3 vector (GenePharma), while pEX‐3 empty vector was used as the negative control (NC). MiR‐181a‐5p mimic, miR‐181a‐5p inhibitor, miR‐181a‐5p mimic control and miR‐181a‐5p inhibitor control were separately transfected into Luva cells using Lipofectamine 2000 transfection reagent (11668027; Invitrogen), followed by extraction of exosomes as needed. YY1 overexpression plasmid and NC were also transfected into HTR‐8/SVneo cells utilizing Lipofectamine 2000 and then co‐cultured with exosomes.
Pex 3 vector
Manufactured by GenePharma
Sourced in China
The PEX-3 vector is a plasmid-based expression system designed for the efficient production of recombinant proteins in various cell lines. It contains an optimized promoter and a multiple cloning site for the insertion of genes of interest. The PEX-3 vector provides a reliable and versatile platform for protein expression studies.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (6 mentions)
Pmirglo dual luciferase vector, by Promega (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Pgl3 basic vector, by Promega (2 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Pex 3, by GenePharma (1 mentions)
Puromycin, by Beyotime (1 mentions)
Opti memtm, by Thermo Fisher Scientific (1 mentions)
Plc5 cir vector, by Geneseed (1 mentions)
Pgl3 basic luciferase vector, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Dual luciferase reporter vector, by GenePharma (1 mentions)
Dual luciferase reporter system, by Promega (1 mentions)
Moflo xdp, by Beckman Coulter (1 mentions)
Si nc, by RiboBio (1 mentions)
Lipofectamine ltx transfection reagent, by Thermo Fisher Scientific (1 mentions)
Interferin, by Polyplus Transfection (1 mentions)
Jetprime reagent, by Polyplus Transfection (1 mentions)
Dual luciferase reporter assay system, by Molecular Devices (1 mentions)
20 protocols using pex 3 vector
1
Exosomal miR-181a-5p Regulation of YY1 Expression
2
Overexpression and Knockdown of Circular RNAs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To construct the overexpression plasmids of circPTPRA, LMNB1, EIF4A3, and FUS, the synthetic human circPTPRA cDNA was cloned into the pLC5‐ciR vector by Geneseed Biotech Co., Ltd., the synthetic human LMNB1, EIF4A3, and FUS cDNAs were cloned into the pEX‐3 vector by GenePharma, and the empty plasmid was used as a control. The siRNA sequences of circPTPRA, LMNB1, EIF4A3, and FUS, as well as the mimics and inhibitor of miR‐140‐5p were designed and synthesized by RiboBio Co., Ltd. LipofectamineTM 3000 (Invitrogen) and Opti‐MEMTM (Gibco) were used as transient transfection reagents according to the manufacturer's instruction. Total RNA was extracted 48 h after transfection. The sequences of siRNAs, sh‐RNAs, plasmids, mimics and inhibitors are shown in Additional file 1 Table S1 .
The overexpression and knockdown lentiviruses of circPTPRA and LMNB1 were constructed by Genechem Co., Ltd. Lentiviral infection was performed according to the manufacturer's instruction. Stable transfected cells were selected by culturing in medium containing 5 μg/mL puromycin (Beyotime), and the expression of circPTPRA and LMNB1 was confirmed by quantitative real‐time PCR (qRT–PCR).
The overexpression and knockdown lentiviruses of circPTPRA and LMNB1 were constructed by Genechem Co., Ltd. Lentiviral infection was performed according to the manufacturer's instruction. Stable transfected cells were selected by culturing in medium containing 5 μg/mL puromycin (Beyotime), and the expression of circPTPRA and LMNB1 was confirmed by quantitative real‐time PCR (qRT–PCR).
3
Dual-Luciferase Assay for RAX2 Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BDNF-AS full-length and RAX2 3'-UTR sequences were amplified by PCR and cloned in the pmirGlo Dual-luciferase Vector (Promega, Madison, USA) to construct luciferase reporter vector (BDNF-AS-Wt and RAX2-Wt) (GenePharma, Shanghai, China). The sequence of putative binding site of BDNF-AS (ALU: 1982–2062bp) and RAX2 (ALU: 2116–2419 bp) was replaced as indicated (BDNF-AS-Mut, RAX2-Mut). The responsive RAX2-binding sites in the DLG5 promotor were predicted by bioinformatics tool JASPAR and were determined by dual-luciferase reporter system. Promoter fragments were subcloned into pGL3-Basic-Luciferase vector (Promega, WI, USA). Human full-length RAX2 was constructed in pEX3 vector (GenePharma, Shanghai, China). The assay was performed 48 h after transfection the indicated constructs into 2.4 × 104 HEK-293T cells per well seeded into 96-well plates. Cells were analyzed by the luciferase assay using the dual-luciferase reporter assay system. The relative luciferase activity was expressed as the ratio of firefly luciferase activity to renilla luciferase activity.
4
Overexpression of NOX4 in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gene sequences coding for NADPH oxidase 4 (NOX4) was cloned into the pEX-3 vector (GenePharma, China). Cells were transfected with pEX-3-NOX4 using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer's protocol. After 6 h transfection, the culture medium was replaced with fresh medium for additional 48 h. The efficiency of overexpression was confirmed by Western blot.
5
Characterizing MAFF Binding Sites in Gene Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The putative binding sequence of MAFF in LINC00673 and the mutant sequence were synthesized and cloned into the pmirGLO Dual-Luciferase vector (Promega, Madison, WI, USA). Wild-type pmirGLO-MAFF or MAFF mutant reporter plasmid and shLINC00673 or shNC were co-transfected into HEK293T cells. Luciferase activity was analyzed by the Dual-Luciferase reporter assay system (Promega), which was recorded as the ratio of firefly luciferase activity to luciferase activity.
For the putative binding site of MAFF in ZO-1, occludin, and the claudin-5 promoter region, we identified the predictor using the Dual-Luciferase reporter assay. The fragments of ZO-1, occludin, and claudin-5 promoters were amplified from human geneomic DNA, and then construct the sequence into the pGL3-Basic vector (Promega). Human full-length MAFF was constructed into the pEX3 vector (GenePharma). The luciferase activity of these binding sites was measured as above.
For the putative binding site of MAFF in ZO-1, occludin, and the claudin-5 promoter region, we identified the predictor using the Dual-Luciferase reporter assay. The fragments of ZO-1, occludin, and claudin-5 promoters were amplified from human geneomic DNA, and then construct the sequence into the pGL3-Basic vector (Promega). Human full-length MAFF was constructed into the pEX3 vector (GenePharma). The luciferase activity of these binding sites was measured as above.
6
Dual-luciferase reporter assay for microRNA targeting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The theoretical binding sequence of miR-346(or miR-425-5p)in MIR17HG gene and its mutant sequence were amplified by PCR, synthesized and cloned into a pmirGlo Dual-luciferase vectors (Promega, Madison, WI, USA) to construct dual luciferase reporter vector (GenePharma). HEK293T cells were seeded in 96-well plates and cotransfected with wildtype pmirGLO-MIR17HG (or MIR17HG mutant) reporter plasmid and pre-miR-346 (or pre-miR-425-5p) or pre-NC. Through the Dual-Luciferase Reporter System (Promega), luciferase activity was measured 48 h after transfection. The 3’-UTR sequence of TAL1 containing the putative miR-346(or miR-425-5p)binding sites and their mutant sequences were cloned into Dual-luciferase vectors. The transfection procedure and Luciferase activities measurement were performed similarly as described above.
The MIR17HG and DEC1 promoter regions were amplified from human genomic DNA by PCR. In addition, putative TAL1 binding sites in the PCR conducts were deleted one by one. The PCR products were subcloned into the pGL3-Basic vector (Promega). Human full-length TAL1 gene was constructed in pEX3 vector (GenePharma). Firefly luciferase activity was normalized to renilla luciferase activity for each individual analysis.
The MIR17HG and DEC1 promoter regions were amplified from human genomic DNA by PCR. In addition, putative TAL1 binding sites in the PCR conducts were deleted one by one. The PCR products were subcloned into the pGL3-Basic vector (Promega). Human full-length TAL1 gene was constructed in pEX3 vector (GenePharma). Firefly luciferase activity was normalized to renilla luciferase activity for each individual analysis.
7
Regulation of EPHB4 by miR-130-3p
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We transfected inhibitors and mimics of miR-130-3p using Lipofectamine 3000, which was sourced from Life Technologies. The concentration of the solution was set at 20 nM according to the instructions from the manufacturer. Based on the expression of green fluorescent protein (GFP), we harvested and cultured cells after sorting lentivirus-infected cells with MoFlo XDP (Beckman, USA) for subsequent functional studies. In addition, to explore whether miR-130-3p could regulate EPHB4 manifestation by directly binding to the established 3′ UTR sequence, we established luciferase reporter plasmids that had binding sites or wild-type for EPHB4. To induce the overexpression of circ-EPHB4, we cloned the coding sequence into the pEX-3 vector (Shanghai Gene Pharma Co., Ltd.), respectively. We transfected the above vectors with Lipofectamine® 3000 (Invitrogen, Thermo Fisher Scientific, USA) based on the instructions of the manufacturer. Following a 48 hour transfection, the efficiency of each group of cells was identified using qRT-PCR.
8
Overexpression and Knockdown of circGSE1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To overexpress circGSE1, the circGSE1 overexpression vector was generated by pEX-3 vector (GenePharma, China). To knock down circGSE1, three siRNAs targeting circGSE1 (si-1, si-2, si-3) and a si-NC were synthesized by RiBoBio (Guangzhou, China). The vimentin overexpression plasmid, si-Vimentin, miR-138-5p mimics and inhibitor were purchased from RiBoBio (Guangzhou, China). Transfection of oligonucleotides and plasmids were performed with Lipofectamine 2000 (Invitrogen, USA).
9
HIF-1α Binding Sites in GAPLINC Promoter
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
By screening the promoter region of GAPLINC, two binding motifs (CACGC; Ren et al., 2014 (link) and ACGTG) of HIF-1α were found. To determine the responsive HIF-1α-binding sites in the human GAPLINC promoter, promoter activities were measured using Dual-Luciferase Reporter Assay System. Human full-length HIF-1α sequence was cloned into pEX3 vector (GenePharma). pEX3-HIF-1α and its empty vector were transfected into MKN45 and SGC7901 cells. Renilla promoters were co-transfected as an internal control. Firefly luciferase activity was normalized to renilla luciferase activity for each individual analysis.
10
Circularizing CLK3 and Regulating miR-320a
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Oligonucleotides and plasmids of circCLK3, miR-320a, and FoxM1 were designed and synthesized. The full length of circCLK3 was cloned into pEX-3 vector (Shanghai GenePharma CO. Ltd). Three siRNAs target circCLK3 were synthesized by RiBoBio (Guangzhou, China) as follows: si-circ-1, target: 5′-GGCCTTGTGTGAGCCACCA-3′; si-circ-2, target: 5′-ATAGGCCTTGTGTGAGCCA-3′; si-circ-3, target: 5′-GCCTTGTGTGAGCCACCAT-3′. The mimics and inhibitor of miR-320a were purchased from RiBoBio. A si-FOXM1 target: CTCTTCTCCCTCAGATATAdTdT. The oligonucleotides and plasmids were transfected into cells by LipofectamineTM 2000 (Invitrogen, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions.
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!