Cfx384 multiplex real time fluorescence quantitative pcr instrument

Nuciferine was bought from MedChemExpress Co., Ltd. (Shanghai, China, purity > 98%). Standard EGCG was obtained from Beijing Solarbio Technology Co., Ltd. (Beijing, China, purity > 98%). Chitosan of medium molecular weight was purchased from Sigma–Aldrich (St. Louis, MO, USA, purity > 75%). Lipo3000 transfection reagent was acquired from Thermo Fisher Co., Ltd. TRIzol^® Plus RNA Purification Kit and Transcriptor First Strand cDNA Synthesis Kit were bought from Thermo Fisher (Waltham, MA, USA). Dual-luciferase reporter assay system was purchased from Promega Co., Ltd. (Madison, WI, USA).
The following instruments were used in this study: Zetasizer Nano ZS90 (Malvern Instruments Co., Ltd., Malvern, UK), MIKRO 22R centrifuge (Kirchlengern, NRW, Germany), OLYMPUS BX60 fluorescent microscope camera (OLYMPUS Corporation, Tokyo, Japan), transmission electron microscope (TEM) (Hitachi, Tokyo, Japan), and CFX384 multiplex real-time fluorescence quantitative PCR instrument (Bio-Rad, Hercules, CA, USA).

The expression of the DEGs was analyzed by RT-qPCR using the CFX384 multiplex real-time fluorescence quantitative PCR instrument (Bio-Rad, Berkeley, CA, USA) platform. First-strand cDNA was synthesized from 300 ng of total RNA using Script cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) in a 4 μL total volume. The resulting cDNA was ten-fold diluted and used in 20μL RT-qPCR mixture as follows: Power SYBP Green Master Mix, 10 μL; gene-specific upstream and downstream primers (10 μmol/L), 0.5 μL; sterile water, 8 μL; and cDNA template, 1 μL. The total reaction volume was 20 μL. The RT-qPCR reaction conditions were as follows: initial denaturation at 95 °C for 1 min, subsequent denaturation of 40 cycles of 95 °C for 15 s and annealing at 63 °C for 25 s. Each sample was evaluated independently three times. Relative gene expression analysis for each gene was determined using the 2^−△△Ct method. ACTB was the internal reference gene. All primers were designed using Primer-BLAST in the NCBI database (https://www.ncbi.nim.nih.gov/tools/primer-blast/ accessed on 1 October 2022). Data on the expression of genes of interest are shown in Supplementary Table S2.