Oligo dt primer

Total RNA from cells was isolated with Trizol reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions. In brief, total RNA (2 μg) was used to synthesize the first strand cDNA with oligo (dT) primer (Bioneer, Daejeon, Korea) and Moloney murine leukemia virus reverse transcriptase (Promega, Madson, WI, USA). The synthesized cDNA was subjected to the PCR-based amplification. The PCR products were detected on 2% agarose gels. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was measured as an internal control. The following primers were used: mGAPDH forward, 5′-AGGCCGGTGCTGAGTATGTC-3′, reverse, 5′-TGCCTGCTTCACCACCTTCT-3′; mFGF21 forward, 5′-ACAGATGACGACCAAGACACTG-3′, reverse, 5′-GTCCTCCAGCAGCAGTTCTC-3′; mPERK forward, 5′-ACTGTTGGCTGGCTCTCACT-3′, reverse, 5′-TGCCTTCGGGGTTAGTTATG-3′; mATF4, forward, 5′-GCAAGGATGCCTTTTC-3′, reverse, 5′-GTTTCCAGGTCATCCATTCG-3′; mNRF-1 forward, 5′-CTCCAAACCCAACCCTGTCT-3′, reverse, 5′-TGGTGGCCTGAGTTTGTGTT-3′; and mTFAM forward, 5′-CAGCCAGGTCCAGCTCACTA-3′, reverse, 5′-ATTAGGAGGGTCTCGCTCCA-3′.

Total RNA was isolated from about 1 × 10⁶ IPEC-J2/well using a TRI-reagent^™ solution (ThermoFisher; Carlsbad, CA, USA) according to the manufacturer’s instructions and reverse-transcribed to generate complementary DNA (cDNA) using oligo-dT primers (Bioneer; Daejeon, Korea); purity (260/280 nm ratio) and concentration (at 260 nm) were assessed using a BioSpectrometer^® (Eppendorf AG, Hamburg, Germany). RNA samples were DNAse-treated (Merck; Darmstadt, Germany) and 1 µg/20 µL was reverse-transcribed using a HiScript III Rt SuperMix (Vazyme Biotech Co.; Nanjing, China). RT was performed using a StepOne^™ thermocycler (Applied Biosystems, StepOne software v. 2.3) and, according to the manufacturer’s instructions, under the following thermal conditions: 2 min at 45 °C, 15 min at 37 °C, followed by 5 s at 85 °C. The cDNA samples were stored at − 20 °C.

Total RNA was isolated from the cells by using Trizol (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized using oligo dT primers and M-MLV reverse transcriptase (Promega Corporation, Madison, WI, USA) and oligo dT primers (Bioneer, Daejeon, Republic of Korea). Relative mRNA expression was quantified via real-time PCR using SYBR Green master mix (Bioneer). In quantitative real-time PCR, GAPDH was used for normalization following the 2^−ΔΔCT method.

Total RNA was isolated using the AccuPrep® RNA Extraction Kit (Bioneer Corp., Korea) and the cDNA synthesized from 1 μg of total RNA using oligo (dT) primers (Bioneer Corp., Korea) and the RocketScript^TM Reverse Transcriptase Kit (Bioneer Corp., Korea). qRT-PCR was performed using ExcelTaq 2X Q-PCR Master Mix (SMOBiO, Taiwan) and the CFX96^TM Real-Time System (Bio-Rad, United States). The cycling conditions were as follows: 95°C for 3 min followed by 40 cycles at 95°C for 15°s, 60°C for 30°s, and 72°C for 30°s. The primer sequences are given in Supplementary Table S1 All reactions were run in triplicate, and data were analyzed using the 2^−ΔΔC_T method (Livak and Schmittgen, 2001 (link)). The internal standard was GAPDH. Significance was determined by the Student’s t-test with GAPDH-normalized 2^−ΔΔC_T values (Livak and Schmittgen, 2001 (link)).

Total RNA was isolated from an 8-well pool using a EuroGold Trifast™ kit solution (Euro-clone, Milan, Italy) according to the manufacturer’s instructions and reverse-transcribed to generate complementary DNA (cDNA) using oligo-dT primers (Bioneer; Daejeon, Korea); purity (260/280 nm ratio) and concentration (at 260 nm) were assessed using a BioSpectrometer® (Eppendorf AG, Hamburg, Germany). RNA samples were treated with DNAse (Merck), and 1 µg/20 µL was reverse-transcribed using HiScript® III RT SuperMix (Vazyme Biotech Co.; Nanjing, China). RT was performed using a StepOne™ thermocycler (Applied Biosystems, StepOne™ software v.2.3) according to the manufacturer’s instructions under the following thermal conditions: 2 min at 45 °C, 15 min at 37 °C, followed by 5 s at 85 °C. The cDNA samples were stored at − 20 °C.

Dulbecco's modified Eagle's medium (DMEM) and foetal bovine serum (FBS) were obtained from WelGene (Daegu, Korea). Streptomycin and penicillin were obtained from Lonza (MD, USA). TRI reagent solution (AM9738) and SYBER green master mix were obtained from Applied Biosystems/Ambion (Warrington, UK). Oligo(dT) primers, RT premix, and PCR premix were obtained from Bioneer Co. (Daejeon, Korea). iNOS, COX-2, TNF-α, IL-1β, IL-6, Leptin, adiponectin, RGS5, and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers were obtained from Bioneer Co. (Table 1) (Daejeon, Korea). Total protein lysis buffer (PRO-PREP) and the PRO-Measure protein assay kit were obtained from iNtRON Biotechnology (Seoul, Korea). LPS (Escherichia coli 055:B5) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) were purchased from Sigma (St. Louis, MO, USA). All other reagents and chemicals were obtained from Sigma Aldrich (St. Louis, MO, USA).

Total RNA was extracted from leaves of C. annuum ‘Bukang’ and silenced N. benthamiana plants using GeneAll^RHybrid-R^™ (Gene All Biotechnology, Seoul, South Korea) according to the manufacturer’s protocol. First-strand cDNA was synthesized from 4 μg total RNA using M-MLV reverse transcriptase (Promega, Madison, WI, USA) and oligo-(d)T primers (Bioneer, Daejeon, South Korea) according to the manufacturer’s protocol. For VIGS, the expression of candidate genes was analyzed by semi-quantitative RT-PCR and real-time PCR using gene-specific primers (S1 Table). For the semi-quantitative RT-PCR, Actin transcript was used as an endogenous control [25 (link)]. The real-time PCR was performed using a Lightcycler^®480 instrument (Roche, Switzerland). Thermal cycling was as follows: denaturing at 95°C for 5 min, followed by 45 cycles of denaturing for 10 s, annealing at 60°C for 20 s and extension at 72°C for 15 s.