Tangeretin Transcriptome Analysis in HepG2 Cells

RNA preparation and microarray analysis were carried out as previously described [70 (link)]. Briefly, HepG2 cells were treated with vehicle (0.1% DMSO) or tangeretin (40 μM) for 24 h, and total cellular RNA was isolated using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. RNA concentration and purity were measured and verified for acceptable quality. RNA integrity was analyzed using the Agilent RNA 6000 Nano assay (Agilent Technology, Inc., Santa Clara, CA, USA) and an RNA integrity number (RIN) value >6. Amino allyl antisense RNA (aa-aRNA) was produced by Eberwine-based amplification with an Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion, CA, USA). The aRNAs were labeled with Cy5 fluorescent dye for hybridization with Human Whole Genome One Array Version 7.1 (HOA 7.1, Phalanx Biotech Group, Hsinchu, Taiwan). The fluorescence intensity of each spot was analyzed by GenePix 4.1 (Molecular Devices, Sunnyvale, CA, USA). Genes with expression differences at a p value < 0.05 were identified as DEGs.