Total rna extraction kit

Total RNA was extracted from the leaves of the wild-type and dye1-1 plants using a total RNA extraction kit (RBC Bioscience; http://www.rbcbioscience.com/, last accessed 24 December 2017). First-strand cDNA was synthesized from 500 ng total RNA using ReverTra ACE qPCR RT Master Mix with gRNA Remover (TOYOBO; http://www.toyobo.co.jp/, last accessed 24 December 2017). The transcript level was determined by quantitative RT-PCR using a KAPA SYBR FAST qPCR kit (KAPA Biosystems; http://www.kapabiosystems.com/, last accessed 24 December 2017) and a Rotor-Gene Q real-time PCR cycler (Qiagen; http://www.qiagen.com/, last accessed 24 December 2017). The primers used for amplification are listed in Supplementary Table S1 at JXB online.

Total ribonucleic acid (RNA) was isolated from the excised pinna from one ear per mice at 24-h post-challenge using a Total RNA Extraction Kit (RBC Bioscience, New Taipei City, Taiwan). Complementary deoxyribonucleic acid (cDNA) was then synthesized from 1 μg of total RNA using an iScript™ Select cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Real-time polymerase chain reaction (real-time PCR) analysis was performed using a LightCycler® 480 Instrument II (Roche Applied Science, Penzberg, Germany). The final reaction mixture in each reaction tube consisted of 0.5 μL of cDNA; 0.4 μL of sense and antisense primer solutions, including IFN-γ (forward: 5′-AACGCTACACACTGCATCT-3′, reverse: 5′-TGCTCATTGTAATGCTTGG-3′) and β-actin (ACTB, forward: 5′-ATGGATGACGATATCGCT-3′, reverse: 5′- ATGAGGTAGTCTGTCAGGT-3′) (Integrated DNA Technologies, Coralville, IA, USA); 10 μL of KAPA SYBR® Fast qPCR Master Mix (2x) Kit (Kapa Biosystems, Wilmington, MA, USA); and, 8.7 μL of sterile Milli-Q® water (Merck). The conditions for reverse transcription and the PCR steps were according to the manufacturer’s instructions. The normalization and quantification of mRNA expression was performed using LightCycler® 480 software (Roche Applied Science, Rotkreuz, Switzerland).

After 28 days of induction, total RNA was isolated from the cells by total RNA extraction kit (RBC Real Genomics, RBC Bioscience, Taipei, Taiwan) according to the manufacturer’s instructions. Then, RNA was reverse-transcribed in the presence of oligo-dT primer for complementary DNA (cDNA) synthesis by iScript^™ Reverse Transcription Supermix for RT-qPCR (BioRad, Hercules, CA, USA). The expression of several genes was assessed by using Light Cycler^® 480 (Roche Diagnostics, Basel, Switzerland) and KAPA SYBR-Green PCR Master mix (Applied Biosystems, Carlsbad, CA, USA). The primers used are shown in Table 1. Melting curve analysis was undertaken to determine the specificity of the primers. Gene expression was normalized to the reference gene GAPDH and calculated as the relative expression compared to control cells. The qPCR analyses were performed three times.