Spectramax id5 multi mode

First, 20 μL of 10 μM synthetic DNA, PCR products or gDNA at different concentrations were added to 2.7 μL of a CeO₂ NP stock solution (2.5 wt.% colloidal dispersion in 0.4 M sodium acetate buffer), and to this solution, 22.3 μL of 0.4 M sodium acetate buffer (pH 3.7) and 1 μL of deionized water were added. After incubation for 5 min, to the solution was added 50 μL of 1× TMB substrate solution and 4 μL of 10 mM PPi, which was then incubated for 30 min to develop the colorimetric signal. Not only PPi, but also other substances such as dNTP and rNTPs (4 μL, 10 mM) were tested to evaluate their enhancement effect on the CeO₂ NP-catalyzed oxidation reactions. After centrifugation at 5900× g for 30 s to separate CeO₂ NPs from the reaction solution, the colorimetric signal of the supernatant was measured at a wavelength of 650 nm using a microplate reader (Spectramax iD5 multi-mode; Molecular Devices).

The wild type (WT) and mutated (MUT) lncRNAs containing target miRNA binding sites were synthesized and cloned into the pGreenII 0800-miRNA reporter vector, yielding the lncRNA-WT and lncRNA-MUT vectors, respectively. The miRNA was amplified and cloned into the pCXUN overexpression vector, yielding the miRNA-OE vector. The miRNA-OE/lncRNA-WT, miRNA-OE/lncRNA-MUT, empty vector (PCXUN)/lncRNA-WT, and empty vector (PCXUN)/lncRNA-MUT pairs were co-transfected into rice protoplast cells, which were prepared following the method of Wu et al. (Wu et al., 2017 (link)). After 16 h of transfection, luciferase activities were evaluated with a dual-luciferase reporter assay system (Promega) and a SpectraMax iD5 multi-mode microplate reader (Molecular Devices). In this study, two lncRNA-miRNA interaction pairs (XLOC_042442-miR1846c and XLOC_028297-miR530) were selected to perform the assay (Supplementary Table 1).