Accupower 2x greenstar qpcr master mix

The cDNA samples were diluted 5 times using nuclease-free water and then 10 ng cDNA was used for each qRT-PCR analysis. The AccuPower 2X GreenStar qPCR MasterMix (Bioneer, Daejeon, Korea) and the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) were used for the qRT-PCR analysis. Each 20-µL reaction comprised 5 μL SYBR mix, 1 μL cDNA, 1 μL of each primer (10 μM), and 12 μL ddH₂O. The Brassica ERF1 gene was used as an internal control for normalizing expression levels (Table S17). The PCR program was as follows: 94 °C for 5 min, 40 cycles of 94 °C for 15 s, 62 °C for 20 s, and 72 °C for 20 s. A melting curve analysis was performed at the end of the qRT-PCR analysis to confirm gene-specific products were amplified. The qRT-PCR was completed using three technical replicates and relative gene expression levels were calculated according to the 2^−ΔΔCt method [84 (link)]. The heat map showed expression patterns of 51 BrBBX genes in different organs and during various developmental stages using available transcriptome data [51 (link)]. The relative expression data shown in the heat map were generated using the pheatmap package of R.

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.

Cellular RNA was isolated using the NucleoSpin^® RNA plus kit (Macherey-Nagel, Düren, Germany). To prepare the cDNA, reverse transcription was performed using a ReverTra Ace^® qPCR RT Master Mix (cDNA kit) (TOYOBO, Osaka, Japan). AccuPower^® 2X Greenstar qPCR Master Mix (Bioneer, Daejeon, Korea) was used to amplify the pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6). The primer sequences used are shown in Table 2.