Accupower 2x greenstar qpcr master mix

RAW 264.7 cells (6 × 10⁵ cells/well) were plated in six-well plates and then stimulated with or without LPS (1 μg/mL) for 1 h. After one hour, the cells were treated with flavonoids (2.5 or 5 µg/mL) and kept for 24 h at 37 °C. Total RNA was extracted by the Trizol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and the quality of the RNA was confirmed on 1.5% agarose gel by studying the extent to which RNA bands were intact, and the quantification of the RNA was done by the Biospectrometer (Eppendorf, Germany). RNA was converted into cDNA by iScript™ cDNA synthesis kit (Biorad Laboratories, Inc., Hercules, CA, USA) following the user guidelines. Then, cDNA was diluted 10-fold and used as the template for qRT-PCR analysis. Total of 20 μL of PCR mix comprising cDNA (2 μL), primer (1.5 μL), 10 μL of AccuPower^® 2X GreenstarTM qPCR Master mix (Bioneer, Daejeon, South Korea), and ddH₂O (6.5 μL) was used. All reactions were performed in 96-well plates using Bio-Rad CFX 96 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The primer sequences for TNFα, IL-1β, and β-actin were obtained from Kim et al. [42 (link)]. β-actin was used as an internal control to normalize, and transcripts changes were measured using the 2^−ΔΔCT method. The experiment was performed in triplicate.

HT-22 cells were seeded in 6-well plates. The cells were treated with TLE for 12 h before harvest. Total RNA was extracted by Trizol reagent and RNA concentration was measured using a Nanodrop spectrometer (Thermo Scientific, Rockford, IL, USA). RNA was converted to complementary DNA (cDNA) by reverse transcription using AccuPower RT Premix kit (Bioneer, South Korea). The cDNA was used as a sample for the real-time PCR step. Real-time PCR assay was performed using AccuPower 2X GreenStar^TM qPCR Master Mix (Bioneer, South Korea) in Exicycler^TM 96 (Bioneer, Daejeon, South Korea) using gene-specific primers (Table 1) [22 (link),23 (link)]. The mRNA expression level was calculated by the delta-delta Ct method with β-actin as an internal control.

The 3T3-L1 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM), glutamine, penicillin-streptomycin, fetal bovine serum (FBS), bovine calf serum (BCS), and TRIzol reagent were obtained from Invitrogen (Carlsbad, CA, USA). The cell count kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). An assay kit for TG was obtained from Asan Pharmaceutical Co. (Seoul, Korea). The GPDH activity assay kit was procured from Takara (Kyoto, Japan). The bicinchoninic acid (BCA) protein assay kit was obtained from Thermo Scientific (Pittsburgh, PA, USA). The Puregene DNA isolation kit was purchased from Qiagen (Chatsworth, CA, USA). M-MLV reverse transcriptase and AccuPower® 2X GreenStarTM qPCR Master Mix were purchased from Bioneer (Daejeon, Korea). The AMPK Kinase Assay kit was purchased from Cyclex (Nagano, Japan) and Isorhamnetin was obtained from Sigma Co. (St Louis, MO, USA)