Investigating mRNA Expression Modulation

For mRNA expression analysis, ARPE-19 cells (1 × 10⁵ cells/well) were seeded in 12-well plates and incubated. After incubation, ARPE-19 cells were pretreated with the indicated concentrations of AB_SH (10 and 100 μg/mL) or fucoidan (10 and 50 μg/mL) for 1 h and then incubated with 50 ng/mL TNF-α for 1 h. The cells were then harvested, and total RNA was extracted with TRIzol^® reagent (Thermo Fisher Scientific), according to the manufacturer’s protocol. The isolated RNA concentration was measured using a OPTIZEN NanoQ Lite (KLAB, Daejeon, Korea). Complementary DNA (cDNA) is (1 μg) was synthesized using a RevertAid RT Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative real-time PCR (real-time qPCR) was performed using AccuPower^® 2X GreenStar™ qPCR Master Mix (Bioneer, Daejeon, Korea), according to the manufacturer’s instructions. Each 20 μL real-time qPCR reaction contained an amount of cDNA equivalent to 1 μL of total RNA (20 ng), 1 μL of the forward and reverse primer each (10 μM), 10 μL of the 2X GreenStar Master Mix, and 7 μL of RNase-free water. 18S rRNA was used as a reference gene for the normalization of all samples, and the gene expression levels were calculated using a previously reported method (23 (link)). All primers used are listed in Supplementary Table 1.