The interactions between miR-27b-3p and circRANBP17 or KDM1A were predicted by starbase (http://starbase.sysu.edu.cn/agoClipRNA.php? source = mRNA ) and DIANA tools (http://carolina.imis.athena-innovation.gr ), respectively. The sequences of circRANBP17 and KDM1A 3′-UTR were amplified and then inserted into psiCHECK2 vector (Hanbio Biotechnology (Shanghai, China), named as circRANBP17 WT and KDM1A 3′-UTR WT, respectively. The miR-27b-3p mimics (miR-NC) and luciferase reporter (circRANBP17 WT, circRANBP17 MUT, KDM1A 3′-UTR WT or KDM1A 3′-UTR MUT) were co-transfected into SK-N-SH and IMR-32 cells. The luciferase activity was detected using Dual-Lucy Assay Kit (Solarbio).
Psicheck 2 vector
Manufactured by Hanbio Biotechnology
Sourced in China
The PsiCHECK-2 vector is a dual-luciferase reporter plasmid. It contains two luciferase genes: Renilla luciferase and firefly luciferase. The Renilla luciferase gene serves as the experimental reporter, while the firefly luciferase gene functions as the internal control.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (5 mentions)
Dual luciferase reporter gene assay system, by Promega (4 mentions)
Dual luciferase reporter assay kit, by Promega (3 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Dual lucy assay kit, by Solarbio (1 mentions)
High glucose dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
P atm, by Abcam (1 mentions)
P ampkα, by Abcam (1 mentions)
Entilink 1st strand cdna synthesis kit, by Elk Biotechnology (1 mentions)
Enturbo sybr green pcr supermix kit, by Elk Biotechnology (1 mentions)
P h2ax, by Abcam (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Abcam (1 mentions)
Dual luciferase assay kit, by Promega (1 mentions)
Psicheck 2 vector, by Promega (1 mentions)
Dual luciferase reporter assay system, by Hanbio Biotechnology (1 mentions)
Fast site directed mutagenesis kit, by Tiangen Biotech (1 mentions)
Hblumi dual luciferase reporter assay kit, by Hanbio Biotechnology (1 mentions)
26 protocols using psicheck 2 vector
1
Regulation of circRANBP17 and KDM1A by miR-27b-3p
2
miRNA-Mediated Regulation of HOTAIR and ATG12
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Partial sequences of HOTAIR and ATG12 3′-untranslated region (3′-UTR) containing predicted miR-93 target sites were subcloned into psiCHECK-2 vector by Hanbio Biotechnology Co., Ltd (Shanghai, China), to produce HOTAIR-Wt reporter and ATG12-Wt reporter, respectively. Also, HOTAIR-Mut and ATG12-Mut reporters containing mutant miR-93 binging sites were constructed by Hanbio Biotechnology Co., Ltd. Then, cells were co-transfected with corresponding reporter vector and miR-93 mimic or miR-NC. Forty-eight hours later, cells were collected and lysed for luciferase reporter assay using a dual luciferase reporter assay system (Promega, Madison, WI, USA).
3
Molecular Mechanisms of DOX-Induced DNA Damage
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DOX was provided by Beijing Huafeng United Technology (Beijing, China). Antibodies against GAPDH, ATM, p-ATM, H2AX, p-H2AX, YES1, p-YES1, p53, p-p53, AMPKα, p-AMPKα were purchased from Abcam (Cambridge, UK). Dulbecco’s modified Eagle’s high glucose medium and Dulbecco’s modified Eagle’s medium were provided by GIBCO (Grand Island, New York, NY, USA). Dual-Luciferase Reporter Assay System Kit (Thermo Fisher Scientific, Waltham, MA, USA) and pSI-CHECK2 vector were provided by Hanbio Biotechnology (Wuhan, China). EntiLink™ 1st Strand cDNA Synthesis Kit and EnTurbo™ SYBR Green PCR SuperMix Kit were purchased from ELK Biotechnology (Wuhan, China).
4
Validating miR-552-3p Binding Sites
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Wild type (WT) and mutant type (MUT) of LINC00261 and DIRAS1 fragments containing the binding sites of miR-552-3p were constructed and cloned into the psiCHECK-2 vector (Hanbio Biotechnology Co., Ltd, Shanghai, China). Next, the constructed dual-luciferase reporter plasmids were co-transfected with mimic-NC or miR-552-3p-mimic into 293T cells. After 48 h, the cells were collected and evaluated using a dual-luciferase reporter gene assay system (Promega, Madison, WI, USA).
5
miR-552-3p Binding Site Validation
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Wild type (WT) and mutant type (MUT) of LINC00261 and DIRAS1 fragments containing the binding sites of miR-552-3p were constructed and cloned into the psiCHECK-2 vector (Hanbio Biotechnology Co., Ltd, Shanghai, China). The constructed dual-luciferase reporter plasmids were co-transfected with mimic-NC or miR-552-3p-mimic into 293T cells. After 48 h, cells were collected and evaluated using the dualluciferase reporter gene assay system (Promega, Madison, WI, USA).
RNA pull-down assay TE-1 cells were digested using trypsin and lysed in RNA-binding protein immunoprecipitation (RIP) lysis buffer. The lysis buffer was divided into several aliquots and frozen at -80℃. Next, 1 µg biotin-labeled RNA and 500 µL structure buffer were added into EP tubes using the RNA-protein magnetic bead sedimentation kit (Pierce, Rockford, IL). Cells were immersed in water at 95℃ for 2 min, soaked in ice for 3 min, cultured with 50 µL fully resuspended beads at 4℃ overnight with the supernatant discarded, centrifuged at 12 g for 3 min, rinsed 3 times with 500 µL RIP cleaning solution and mixed with 10 µL cell lysate for 1 h. In addition, protein in the incubated bead-RNA-protein mixture was eluted. Protein concentration was determined using the bicinchoninic acid method. RNA expression was determined by RT-qPCR.
RNA pull-down assay TE-1 cells were digested using trypsin and lysed in RNA-binding protein immunoprecipitation (RIP) lysis buffer. The lysis buffer was divided into several aliquots and frozen at -80℃. Next, 1 µg biotin-labeled RNA and 500 µL structure buffer were added into EP tubes using the RNA-protein magnetic bead sedimentation kit (Pierce, Rockford, IL). Cells were immersed in water at 95℃ for 2 min, soaked in ice for 3 min, cultured with 50 µL fully resuspended beads at 4℃ overnight with the supernatant discarded, centrifuged at 12 g for 3 min, rinsed 3 times with 500 µL RIP cleaning solution and mixed with 10 µL cell lysate for 1 h. In addition, protein in the incubated bead-RNA-protein mixture was eluted. Protein concentration was determined using the bicinchoninic acid method. RNA expression was determined by RT-qPCR.
6
miR-200a-3p Regulation of TXNIP 3'UTR
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The ability of miR-200a-3p to interact with the TXNIP 3′-untranslated region (UTR) was predicted using the TargetScan (http://www.targetscan.org ) and miRDB (http://mirdb.org ) databases. These putative interactions were then tested via a luciferase reporter approach. Briefly, wild-type (wt) and mutant (mut) versions of the TXNIP 3′-UTR were cloned into the psiCHECK-2 vector (Hanbio Biotechnology Co., Ltd., Shanghai, China) to prepare reporter plasmids which were then co-transfected into HEK-293T cells along with miR-200a-3p mimics or corresponding negative control constructs using Lipofectamine 2000 (Invitrogen, USA). At 48 h post-transfection, a Dual-Luciferase assay kit (Promega, USA) was used based on provided directions, with Renilla luciferase being used for normalization purposes.
7
Investigating HSPA5-miR-197-3p Interaction
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The relationship between HSPA5 and miR-197-3p was determined using the dual luciferase reporter assay. HSPA5 containing the predicted binding site (HSPA5-WT) or mutant binding site (HSPA5-Mut) was amplified and cloned into the pSI-Check2 vector (Hanbio, China). The miR-197-3p mimic, NC mimic, and plasmid were co-transfected into HEK-293T cells. At 48 h after transfection of the pSI-Check2 vector, the luciferase activity was measured using the dual luciferase reporter gene assay system (Promega, E1910).
8
Verification of circFkbp5-miR-760-3p-TNF-α Axis
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For verification of the miR-760-3p binding ability to circFkbp5 and TNF-α. The sequences of circFkbp5, TNF-α-3’UTR and their miR-760-3p binding sites mutants were synthesized and cloned to psiCHECK2 vector (Hanbio Biotechnology, Wuhan, China), respectively named as circFkbp5-wt, circFkbp5-Mut, TNF-α-wt, TNF-α-Mut. The laboratory protocol was guided by the manufacturer’s instructions of the Dual-Luciferase Reporter Assay kit (Promega).
9
RPPH1 mRNA-miR-122 Binding Assay
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The binding site region of RPPH1 mRNA and miR-122, including the wild-type (wt) RPPH1-3'-UTR and mutant (mut) RPPH1-3'-UTR, was cloned into the pSI-Check2 vector (HanBio). HCCLM9 cells were co-transfected with the vectors and the miR-122 or NC mimics for 48 h. Next, the supernatants were collected, and the luciferase activity was measured using a dual-luciferase reporter assay system (HanBio).
10
Luciferase Reporter Assay for miRNA Binding
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Sequences for wild-type (WT) or mutant (Mut) NORAD or the 3′-UTR of MTDH containing the predicted binding sites for miR-224-3p were subcloned into a psiCHECK-2 vector (Hanbio Biotechnology, Shanghai, China). The luciferase reporter plasmids were co-transfected into ESCC cells with a miR-224-3p mimic or negative control (NC). After 48 h, the cells were collected and counted. A total of 5 × 105 cells were used to measure luciferase activity. Relative luciferase activity was measured by using an HBLumi Dual-luciferase reporter assay kit (Hanbio Biotechnology) according to the manufacturer’s instructions. Renilla luciferase activity was normalized to firefly luciferase activity. To evaluate the effect of the miR-224-3p mimic on firefly luciferase activity, the miRNA mimic NC group was used as control. All assays were carried out in triplicate. The mutant sequences of NORAD and the MTDH-3′-UTR were created by using the FAST Site-Directed Mutagenesis Kit (Tiangen, Beijing, China).
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