Accupower 2 greenstar qpcr master mix

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, and antibiotics were obtained from Hyclone (Logan, UT, USA). LPS, dexamethasone (DMS), bovine serum albumin (BSA), and dimethyl sulfoxide (DMSO) were purchased from Sigma–Aldrich (St. Louis, MO, USA). ELISA antibody sets were obtained from eBioscience (San Diego, CA, USA). Cell culture dishes and plates were purchased from Sarstedt (Nümbrecht, Germany). A CCK was obtained from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Cell Signaling Technology, Inc. (Boston, MA, USA). An RNA extraction kit was obtained from iNtRON Biotech (Daejeon, Korea). DNA synthesizing kits and an AccuPower^® 2× Greenstar qPCR Master Mix were obtained from Bioneer (Daejeon, Korea). Oligonucleotide primers for real-time RT-PCR were synthesized by Bioneer. ARAE was obtained as a freeze-dried powder from the Korea Plant Extract Bank (Ochang, Korea), dissolved in dimethyl sulfoxide (DMSO) and stored at −20 °C before use. Chemical standards for ARAE (atractylenolide I, atractylenolide III, and atractylodin) were purchased from ChemFaces (Wuhan, China).

Total RNA was extracted from 5-day-old seedlings using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1 μg total RNA using a cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA). To rescue chloroplast-expressed RNA, cDNA was synthesized, using oligo dT and random primers. The cDNA template was analyzed by qRT-PCR using a CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), gene-specific primers (Supplementary Table S1), and AccuPower 2× GreenStar qPCR Master Mix (Bioneer, Daejeon, Republic of Korea). EF1α was used as an internal reference gene for data normalization. The qRT-PCR was performed using the following conditions: 95 °C for 5 min, followed by 45 cycles of 95 °C for 20 s, 58 °C for 20 s, and 72 °C for 20 s. The mean level of expression of each gene was determined, using the comparative Ct method (2^−ΔΔCt). Data and error bars are indicated as means ± standard deviation (SD). The experiments were performed independently three or four times.

Total RNA from Kidney cortex region was isolated using TRIzol reagent (Thermo Fisher Scientific) according to manufacturer’s instructions^{52 (link)}. Double-stranded cDNA synthesis was prepared by using the iScript cDNA Synthesis Kit (BioRad, Hercules, CA, USA). The resulting cDNA was subjected to quantitative real-time PCR using the StepOnePlusTM Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) with AccuPower 2 × Greenstar qPCR Master Mix (Bioneer, Daejeon, Korea) according to the standard protocol. The primer sequences for each genes are as follows: for NQO1, forward, 5′-GCCCGCATGCAGATCCT-3′ and reverse, 5′-GGTCTCCTCCCAGACGGTTT-3′; AhR, forward, 5′-GCAGGATTTGCAAGAAGGAG-3′ and reverse, 5′-ACTGCTGAAAGCCCAGGTAA-3′; NRF2, forward, 5′-GGCCTTTTTTGCTCAGTTTCA-3′ and reverse, 5′-ATGTGGGCAACCTGGGAGTA-3′; HO1, forward, 5′-CAGCCCCACCAAGTTCAAAC-3′ and reverse, 5′-GGCGGTCTTAGCCTCTTCTGT-3′; catalase, forward, 5′-CGACCAGGGCATCAAAAACT-3′ and reverse, 5′-ATTGGCGATGGCATTGAAA-3′; MnSOD, forward, 5′-CCACACATTAACGCGCAGAT-3′ and reverse, 5′-TCGGTGGCGTTGAGATTGT-3′; Gpx, forward, 5′-TCCGGGACTACACCGAGATG-3′ and reverse, 5′-TTCTCCTGGTGTCCGAACTGA-3′. Gene expression were normalized to that of 18S rRNA.