Dual luciferase reporter assay kit

The WT and mutant variants LINC01606 or the 3′‐UTR of the WT and mutant variants SCD1 plasmids were cotransfected concurrently with miR‐423‐5p mimics and NCs after seeded into six‐well plates for 24 h. Luciferase assays were performed using a Dual‐Luciferase reporter assay kit (Beyotime Biotechnology) as described in the protocols of manufacturer after transfection for 48 h. The luciferase activities were measured on the luminometer microplate reader (Thermo Fisher) following the manufacturer's instructions.
For the TOPFlash/FOPFlash reporter assay, cells were cultured in six‐well plate for 24 h and cotransfected with 1 μg of SuperTOPFlash plasmid, SuperFOPFlash plasmid using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, respectively. Meanwhile, 0.5 μg pRL‐TK Renilla control luciferase plasmid was cotransfected to normalise for transfection efficiency. After transfection for 48 h, cells were lysed using lysis buffer, and luciferase activity was assayed in a luminometer using the Dual‐Luciferase reporter assay kit (Beyotime Biotechnology). Topflash and Fopflash luciferase values were normalised to Renilla luciferase activity, and values were normalised to control.

Firstly, the sequence fragment of LDOC1 3′-untranslated region (3′UTR) was artificially synthesized and introduced into the psiCHECK-2 vector (Promega, Madison, WI, USA). The mutation sites in the complementary sequence of seed sequences were designed on the wild type (WT) of LDOC1. Subsequently, all luciferase reporter plasmids, such as LDOC1 3′UTR-WT and LDOC1 3′UTR-mutant (MUT), were obtained. All aforementioned plasmids were co-transfected with miR-4532 mimic (2 nM, Dharmacon, Lafayette, CO, USA) or mimic-NC (2 nM, Dharmacon, Lafayette, CO, USA) into 4 × 10⁵ HEK-293 T cells (CRL-1415, Shanghai Xin Yu Biotech Co., Ltd., Shanghai, China) and CD34⁺ HSCs, respectively. After 48 h, the cells were lysed and the luciferase activity was determined using Dual-Luciferase Reporter Assay kits (RG005, Beyotime Biotechnology, Shanghai, China) in the Glomax20/20 luminometer fluorescence detector (Promega, Madison, WI, USA). The experiment was repeated three times to obtain the mean value [24 (link)].

The target gene of miR-126 was confirmed by Luciferase reporter gene analysis. miR-126 was predicted with the complementary sequence of 3′-UTR of LRP6 by Target scan (http://www.targetscan.org/), and 24-well plate HUVECs were transfected with Lipofectamine 2000, mimic-miR-126, and NC-miR-126 for 18 hours. The luciferase activities were demonstrated through Dual-Luciferase Reporter Assay kits by following the company's instructions (Beyotime, Jiangsu, China), and the values were normalized to Renilla, and the data were measured by fluorescent activity.