Luc pair duo luciferase assay kit

RNA22 (see text footnote 1) and starBase V2.0³ were employed to screen for potential target lncRNAs and genes of miR-210-3p, among which DLEU2L and breast cancer type 2 susceptibility protein (BRCA2) were selected, respectively. Binding sites between miR-210-3p and DLEU2L or the 3′ untranslated region (UTR) of BRCA2, were identified, and wild-type (WT) and mutant (MUT) 3′UTR segments of the binding sites were prepared based on the predicted sequences. Transfection was performed with pmirGLO plasmids (Addgene, Watertown, MA) using the LucPair^TM Duo-Luciferase Assay Kit (LF001, GeneCopoeia, Rockville, MD). After transfected cells were cultured with miR-210-3p mimics or negative control (NC), the signal of firefly luciferase and renilla luciferase was detected using a Glo-MAX 20/20 analyzer.

The full-length human MDR1 3′-UTR containing the predicted miR-298 binding site (445–451 bp) was amplified from the genomic DNA using specific primers (Table 1). The Duo-Luciferase reporter vector pEZX-FR2 (GeneCopoeia Inc., Rockville, MD, United States) was linearized with EcoRI and XhoI restriction enzymes, and the PCR product was cloned into the linearized vector using an EasyGeno Assembly Cloning kit (Tiangen Biotech Co., Ltd., Beijing, China) to create the vector pEZX-MDR1-3′-UTR. The consensus miR-298 binding site was mutated using a QuikChange II site-directed mutagenesis kit (Stratagene, Agilent Technologies, Santa Clara, CA, United States) to construct a clone of the mutant MDR1 3′-UTR, which was referred to as pEZX-MDR1-3′-UTR-mut. All clones were verified by DNA sequencing. For the luciferase reporter assay, HEK293T cells seeded in 24-well plates were transiently transfected with pEZX-MDR1-3′-UTR or pEZX-MDR1-3′-UTR-mut reporter plasmids together with a miR-298 mimic or miR-NC using Lipofectamine iMax (Invitrogen, Carlsbad, CA, United States). Twenty-four hours after transfection, cells were lysed, and their luciferase activities were determined using a Luc-Pair^TM Duo-Luciferase Assay Kit (GeneCopoeia), according to the manufacturer’s instructions.

For luciferase reporter assays, DF-1 cells were seeded in 48-well plates and transfected with Luc2-IRES-Reporter vectors as described in section “Vector construction and RNA oligonucleotides”. After for 48 h, the luminescent values of firefly and Renilla luciferases were detected using the LucPair^TM Duo-Luciferase Assay Kit (GeneCopoeia, Rockville, MD, United States) with a fluorescence/multi-detection microplate reader (US BioTek Laboratories, Shoreline, WA, United States).

A segment of the murine Nrp2 3′-UTR (1,224 bp) containing potential miR-200-binding sites was amplified from 344SQ genomic DNA through PCR and inserted into the pCI-neo-hRL vector. The putative miR-200-binding sites within the Nrp2 3′-UTR were identified using TargetScan. Subsequently, 3′-UTR reporters (500 ng) and pGL3-control (50 ng; Promega) were co-transfected into 293T cells seeded in 24-well plates (1 × 10⁵ cells/well) using the TransIT-X2 transfection reagent (Mirus Bio). The transfections were performed in the presence or absence of miR-200 mimic (50 nM; Bioneer). Two days after transfection, luciferase activity was measured using the Luc-Pair Duo-Luciferase Assay Kit (GeneCopoeia, Rockville, MD, USA).

Due to the large size of the 3′UTR of human IGF1R (7115 bp), analysis was performed on three distinct constructs comprising its full length (see Supplemental Materials). The constructs were inserted into vectors linked to the Firefly luciferase gene in addition to the Renilla luciferase gene for normalization (Genecopoeia, Rockville, MD, USA). Each fragment of the IGF1R 3′UTR contained a unique theoretical interaction site between let-7c-5p and the IGF1R gene. Additional mutant constructs were created through site-directed mutagenesis to assess each potential binding site. Caki-1 cells were grown in CELLSTAR 24-well dishes (Greiner) and, upon displaying 70–80% confluency, cell transfection was performed. At this time, the medium was replaced with Opti-MEM™ Reduced Serum Medium (31985062, Invitrogen), and transfected with a vector (0.2 μg) along with either pre-let-7c-5p or pre-miR-Precursor Negative Control #1 (30 nM), delivered with Endofectin (Genecopoeia). Cells were lysed according to the manufacturer’s protocol 24 h following transfection. Firefly and Renilla luminescence was measured using the Luc-Pair™ Duo-Luciferase Assay kit (Genecopoeia) in the GloMax^® 20/20 Luminometer (Promega).

The wild‐type (WT) enhancer sequence and three mutated sequences were PCR amplified (WT:CTGTCAACCTTGCTTCCTCCCCttcccacgCACCTTCCATGCATGTACACAC, Mut1: CTGTCAACCTTGCTTCCTCCCCttcccgactcaCTTCCATGCATGTACACAC, Mut2: CTGTCAACCTTGCTTCCTCCCCttcccaCACCTTCCATGCATGTACAC, and Mut3: CTGTCAACCTTGCTTCCTCCCCcgtaatacCACCTTCCATGCATGTACACAC) and cloned into a pGL4.24 firefly luciferase reporter plasmid (Promega). These luciferase reporter vectors and Renilla luciferase vector (pRL‐SV40, Promega) were co‐transfected into mouse intestine cells using Lipofectamine 3000 (Life Technologies) according to the manufacturer's instructions. Cell lysates were collected, and luciferase samples were prepared using the Luc‐Pair Duo‐Luciferase Assay kit (Genecopoeia) in 48 h after transfection. Firefly luciferase activities were measured using an FLUOstar optima plate reader (BMG Labtech), and firefly luciferase activity was normalized to Renilla luciferase activity.