Prime q mastermix

Total RNA was isolated using the AccuPrep^® RNA Extraction Kit (Bioneer Corp., Daejeon, Korea), and the cDNA was synthesized from 1 μg of total RNA using oligo (dT) primers (Bioneer Corp., Daejeon, Korea) and the RocketScript^TM Reverse Transcriptase Kit (Bioneer Corp., Korea). qRT-PCR was performed using Prime Q-Mastermix (GeNet Bio, Daejeon, Korea) and the CFX96^TM Real-Time System (Bio-Rad, Hercules, CA, USA). 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 shown in Table S1. All reactions were run in triplicate, and data were analyzed using the 2^−ΔΔCT method [41 (link)]. The internal standard was GAPDH. Significance was determined by the Student’s t-test with GAPDH-normalized 2^−ΔΔCT values [45 (link)].

Total RNA was isolated from A549 cells and accessory lung lobes of K-ras^LA1 mice using the QuickGene RNA kit (Fujifilm's Life Science System, Tokyo, Japan). Reverse transcription was performed using SuPrimedScript RT Premix (GeNet Bio, Cheonan, Korea) for conversion to cDNA. Reverse transcription was carried out at 25°C for 10 min, 37°C for 30 min, and 85°C for 5 min. Quantitative real-time PCR was performed using the CFX96^™ Real-Time System (Bio-Rad, Richmond, CA, USA), and cDNA was amplified using Prime Q-mastermix (GeNet Bio) and specific primers following the method and conditions used for previously described quantitative real-time PCR experiments [45 (link)]. The result was analyzed using Bio-Rad CFX Manager Version 2.1 software (Bio-Rad). Sequences of primers were as follows: human ERGIC3, forward (5′-TCGCTGTGAGAGCTGCTATG-3′) and reverse (5′-C GCACATCTTCACAGGTGTT-3′); human GAPDH, forward (5′-GCCCAATACGACCAAATC -3′) and reverse (5′-ACTCAGCCGCATCTT-3′); mouse ERGIC3, forward (5′-CCACAGTGTACATGAAGGTGGA-3′) and reverse (5′-GAGCTCATACAGCACAAAGACC-3′); mouse Actin, forward (5′-TTTCCAGCCTTCCTTCTTGGGTATG-3′) and reverse (5′-CACTGTGTTGGCATAGAGGTCTTTAC-3′).

Total RNA was purified from 10 flies per each genotype (five males and five females) using Trizol Reagent, according to the manufacturer’s instructions (Thermo Fisher Scientific). cDNA was prepared from DNase I-treated RNA samples using the M-MLV Reverse Transcriptase reagent (Promega) and random hexamers. Diluted cDNA samples were quantitatively analyzed by SYBR Green-based Prime Q-Mastermix (GeNet Bio) and gene-specific primers using the LightCycler 480 real-time PCR system (Roche). To validate the efficiency of transgenic RNA interference, total RNAs from head or body extracts were analyzed similarly.

Total RNA extraction from rice samples and cDNA synthesis were accomplished using the methods described above. Quantitative real-time PCR (qRT-PCR) was performed using a Prime Q-Mastermix (GeNet Bio, Daejeon, Korea) on a Rotor-Gene Q instrument system (Qiagen). For normalization of transcript levels, rice ubiquitin5 (UBQ5) gene (Os01g22490) was used as a reference gene, which expresses stably in rice (Jain et al., 2006 (link)). The _ΔΔCt method was applied to calculate expression levels (Choi et al., 2014 (link)). To ensure primer specificity, we used the data when the melting curve showed a single peak. Primers for qRT-PCR are listed in Supplementary Table 2. The primer sequences for OsCCR19, 20, 21, and UBQ5 were followed by Koshiba et al. (2013 (link)). To assess the expression of OsCCR17, 19, 20, and 21 in different tissues and stress conditions, qRT-PCR analysis was performed on triplicated biological samples, and each sample was analyzed twice for technical replicate. The results represent the mean ± standard deviation. One-way ANOVA and Tukey's HSD post-hoc test for qRT-PCR data were performed and significant differences (p < 0.05) were represented with the letters a and b. All statistical analysis was carried out using SPSS statistics.

Microarray data for OsCHSs at different development stages of rice were downloaded from the Genevestigator plant biology database (https://genevestigator.com/gv/doc/intro_plant.jsp) [44 (link)]. The normalized data were used to generate heatmap expression patterns using the Multi Experiment Viewer program (http://www.tm4.org/mev.html).
cDNAs synthesized from different tissues and developmental stages of rice were used as a template for qRT-PCR using a Prime Q-Mastermix (GeNet Bio, Daejeon, Korea) on an AriaMx real-time PCR system (Agilent, Santa Clara, CA, USA). Transcript levels were normalized by rice ubiquitin 5 (OsUBQ5) transcripts as a control. The ΔCt method was applied to calculate expression levels. We used primers that showed a single peak in melting curve data. The sequences and annealing temperatures of primers for qRT-PCR are listed in Table S3.