Hiscript 2 qrt supermix 2

Total RNA was extracted from Arabidopsis and wheat using Trizol reagent (TaKaRa, 9109, Tokyo, Japan). Total RNA (500 ng) was used to generate cDNA using 5× HiScriptII qRT SuperMixII (R223-01, Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China). Real-time PCR was performed using SYBR qPCR master mix (Q711-02, Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China) with the CFX96TM Real-Time system (Bio-Rad, Hercules, CA, USA). All primers used in this study are listed in Table S1. The expression levels of the target genes were normalized to the expression levels of the internal controls, i.e., TaACTIN in wheat and AtUBC30 in Arabidopsis.

When Bacillus sp. S3 growth reached middle exponential phase, culture aliquots were amended with 0, 100, 200 and 300 μM of Sb(III), respectively. The culture aliquots were withdrawn at different time intervals (0.5, 1, 2 and 4 h) and cells were harvested by centrifugation at 4000×g for 10 min at 4 °C. After quick freezing in liquid nitrogen, the total RNA of each sample was isolated and purified using the E.Z.B.A Bacterial RNA Kit (Omega, Bio-Tek, USA) according to the manufacturer’s instructions. The concentration of RNA samples was measured at the A_260/280 ratio using a NanoDrop ND-1000 Spectrophotometer (BioTek Instruments, Inc., Vermont, USA) and the integrity of the RNA samples was determined by 1.0% agarose gel electrophoresis. First-strand cDNA was synthesized with 2 μg of total RNA in a 20 μL total reaction volume using the 5 × HiScriptII qRT SuperMixII (Vazyme Biotech Co., Ltd., China). RT-qPCR analysis was performed using iCycler iQ Real-time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, USA) with a 20 μL reaction volume and using 2 × ChamQ™ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., China). Primers were designed using Primer Premier 5 software [19 (link)]. Gene expression was calculated by 2^-∆∆Ct method as follows: 2^-∆∆Ct = 2^{-[(CtA-CtB) treated- (CtA-CtB) control]}, where A denotes target gene and B denotes control gene [22 (link)].

Total RNA was extracted from whole dish cells or zebrafish embryos (approximately 10 embryos per group) using RNAiso Plus reagent (Takara, Japan). cDNA was generated using Hiscript II qRT SuperMix II (Vazyme Bio, Nanjing, China). qPCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme Bio, Nanjing, China) on a QuantStudio 1 (Thermo Scientific^TM, Waltham, MA, USA). The fold change of target genes was calculated with the 2^−ΔΔCt method.

On the night before spawning, the paired adult zebrafish are placed separately on both sides of the breeding tank, and the divider is opened at 8:30 a.m. on the spawning day to start mating. One-cell stage embryos were placed on a fluted agarose plate containing a small amount of EM solution. Approximately 1 nL of the mix solution or mRNA was injected per embryo.
Total miRNA was isolated from the 4 dpf (day post fertilization) larvae which expressed red fluorescence using the miRNA Isolation Kit (Tiangen, DP501, China), according to the manufacturer’s protocols. RNA concentration was measured by Nanodrop (Thermo Scientific, Waltham, USA). 1 μg miRNA was reversely transcribed into cDNA with miRNA First-Strand cDNA Kit (Tiangen, KR211, China) and cDNA was quantified by qPCR system (Light-Cycler 96, Roche) with miRNA qPCR Kit (Tiangen, FP411, China).To detect RNA levels, 4 dpf larvae with red fluorescence were selected to isolate total RNA using TRIzol (TARAKA, China), and the RNA was reverse-transcribed into cDNA with HiScript II qRT SuperMix II (Vazyme, China). The mRNA expression levels were quantified by the qPCR system using AceQ qPCR SYBR Master Mix (Vazyme, China). Twenty-five larvae were collected for each experimental condition. Each experiment was repeated three times in biology and technology. The qPCR primers used are listed in the Supplementary Table S1.

Leaves from four uniform seedlings that were planted in a plastic pot (5 cm in diameter and 6 cm deep) containing nutrient soil were pooled for RNA extraction using Trizol reagent (ThermoFisher Scientific, Waltham, MA), and the gDNA wiper Mix (Vazyme) was used to remove DNA. HiScript II qRT SuperMix II (Vazyme) was used to synthesize complementary DNA (cDNA). qRT-PCR was performed using the SYBR Green RT-PCR kit (ThermoFisher Scientific). The normalized expression levels were analyzed as described in Livak and Schmittgen (2001) (link). Three biological replications were performed for muw and wild-type samples respectively. The gene-specific primers used are listed in Table S1.

Total RNA was isolated from yeast cells with a total RNA extraction kit (Tiangen Biochemical Technology Co., Ltd.). We then used qRT-PCR to determine the relative mRNA expression levels of GAP1, ARO8, ARO9, ARO10, or ENO1. The reaction mixture for reverse transcription included 1μg of total RNA, 4 μL of 4 × gDNA wiper Mix, 4 μL of 5 × HiscriptIIqRT SuperMix II, and RNase free ddH₂O (Vazyme Biotech Co., Ltd.). PCR was performed at 50°C for 15 min and 85°C for 5 s. A 20 μL reaction mixture was prepared for each qPCR reaction and contained 10 μL of ChamQ Universal SYBR qPCR Master Mix, gene-specific primers (Table 2), 1 μL of Temple DNA/cDNA, and RNase free ddH₂O. PCR was then performed at 95°C for 30 s, with 40 cycles of 95°C for 10 s and 60°C for 30 s, 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s, using a QuantStudio 3 real-time PCR system (Thermo Fisher Scientific). Delta cycle threshold (ΔC_T) values were calculated by the C_Ts of the target genes minus the C_T of ENO1, which was used as a housekeeping gene. ΔΔC_T values were calculated by ΔC_T values from the experimental samples – the C_T of the control sample. Fold changes were calculated using the 2^–ΔΔCT method (Livak and Schmittgen, 2001 (link)).