Revert aid reverse transcriptase

The total RNA of liver, duodenum, jejunum, and ileum was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The concentration of RNA was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and its integrity verified by electrophoresis on a 1% agarose gel. The cDNA was reverse transcribed from 1 μg of total RNA using the Revert Aid Reverse Transcriptase (Takara, Japan), then used for evaluating gene expression. The primers used for target genes are presented in Table 2. The qPCR was performed in a 10-μL reaction volume including 0.5 μM of each forward and reverse primer, 2 μL of cDNA, 2 μL of DEPC treated water, and 5 μL of SYBR Premix Ex Taq (Takara Bio Inc., Japan). The relative expression levels of genes were performed using the Lightcycler-480II system (Roche Diagnostics GmbH, Mannheim, Germany). The PCR cycling condition was 40 cycles at 94 °C for 40 s, 60 °C for 30 s and 72 °C for 35 s. Zn-Met group served as the control group and ZnSO₄, _feed and ZnSO₄, _water groups served as treatment. The relative expression was expressed using the formula 2^{−(∆∆Ct)}, where ∆∆Ct = (Ct_Target − Ct_β-actin)_treatment − (Ct_Target − Ct_β-actin)_control [16 (link),17 (link)]. Relative expression was normalized and expressed as a ratio to the expression in the Zn-Met group.

Total RNA of mandarin fish brains was extracted with Trizol Reagent (TaKaRa, Tokyo, Japan) according to the manual. The purity and quantity of total RNA were determined using the BioTek Synergy 2 luminometer (BioTek, Winooski, VT, USA), and integrity of total RNA was checked using electrophoresis in 2% agarose gel (Biowest Agarose, Madrid, Spain). The cDNAs were obtained from 1 μg total RNA with the Revert Aid™ Reverse Transcriptase (TaKaRa, Tokyo, Japan) according to the manufacturer’s instructions.

To investigate whether ethylene biosynthesis and signaling were involved in the plant response to Cd stress, the relative expression of genes encoding for ethylene biosynthesis, and of perception and signaling proteins were measured in seedlings that were treated with different concentrations of CdCl₂ for 4 days. Total RNA was isolated from 100 seedling roots using TRIzol solution (Invitrogen, Carlsbad, CA, USA). Two micrograms of total RNA was used for first-strand cDNA synthesis using RevertAid Reverse Transcriptase and Oligo d(T)primers (Takara, Dalian, China). The cDNA yield was measured according to the PCR signal generated from the internal standard, with the housekeeping gene Actin 2 used as the internal control. The primers that were used in the quantitative RT-PCR are listed in Table 1 (Liu et al., 2010 (link); Lin et al., 2013b (link); Li et al., 2014 (link)).
Quantitative RT-PCR was performed using a RealMasterMix kit (Tiangen, Beijing, China) with 25 cycles as follows: 94°C for 30 s, 58°C for 30 s and 72°C for 30 s, followed by 72°C for 10 min gene expression quantifications was performed using the relative 2^-ΔΔCt method (Livak and Schmittgen, 2001 (link)). All experiments were performed in triplicate for each treatment.

Total RNA was extracted using Trizol reagent (TaKaRa, Tokyo, Japan) following the manufacturer's instructions. The extracted RNA was re-suspended in 30 μL RNase-free water and then quantified with a BioTek Synergy™ 2 Multi-Detection Microplate Reader (BioTek Instruments, Winooski, USA) and agarose gel electrophoresis. One microgram of total RNA was synthesized to complementary DNA (cDNA) using Revert Aid™ Reverse Transcriptase (TaKaRa, Tokyo, Japan) according to the manufacturer's instructions. The synthesized cDNA was stored at −20°C.

The total RNA was isolated with the TRIzol reagent (Invitrogen, USA) before being examined with the NanoDrop 2000 (Thermo Fisher, USA). One microgram of total RNA was synthesized to complementary DNA (cDNA) by Revert Aid Reverse Transcriptase (TaKaRa, Tokyo, Japan). For miRNA, the MirX miRNA 1st Strand Synthesis kit (Clontech, USA) was employed for the cDNA synthesis. The expressions of the circRNA, miRNA, and mRNA were assessed through qRT-PCR, which was accomplished with the SYBR qRT-PCR Master Mix (Vazyme, China) or miRNA qRT-PCR SYBR Kit (Clontech), as appropriate. GAPDH and U6 were employed as the internal controls. The primer sequences are shown in in Table 1.Table 1

The sequences of the primers in this study

Primer	Sequences
Circ-NFKB1	Forward: 5′- GAGGATGGGATCTGCACTGT-3'
	Reverse: 5′- GCGAAACCTCCTCTTCCTG-3'
miR-203a-5p	Forward: 5′- GTGAAATGTTTAGGACCACTAG -3′
Collagen II	Forward: 5′- TGAGAGGTCTTCCTGGCAAA -3' Reverse: 5′- ATCACCTGGTTTCCCACCTT -3'
MMP1 MMP13 ERBB4 GAPDH	Forward: 5′- GCCATATATGGACGTT-3' Reverse: 5′- CACTTCTCCCCGAATCGT-3' Forward: 5′- GCCAGATGGGTTTTGAGAC-3' Reverse: 5′-GTGATGCCTGGGGACTGTT-3′ Forward: 5′-TAGACCCGGGAGAAGGAAGAGCATC-3' Reverse: 5′-TCGCCCGGGTTATGACACCACAGTATTCCG-3' Forward: 5'-CCACTCCTCCACCTTTGACG-3'
	Reverse: 5′-CCACCACCCTGTTGCTGTAG-3'
U6	Forward: 5′-GCGCGTCGTGAAGCGTTC-3'
	Reverse: 5′-GTGCAGGGTCCGAGGT-3'