Ethics Statement All experiments involving the handling and treatment of fish in this study were approved by the Animal Care and Use Committee of Heilongjiang River Fisheries Research Institute of Chinese Academy of Fishery Sciences (HRFRI). The methods were carried out in accordance with approved guidelines. Before samples were collected, all the fishes were euthanized in MS222 solution. In addition, we have followed the ARRIVE guidelines (https://arriveguidelines.org).
2.1 Fish source and heat-stress experimental settings
Fishes used in this study were cultured at the Bohai Cold Fish Experimental Station of the Heilongjiang River Fisheries Research Institute. Healthy individuals with body weights of 15 ± 5.0 g were used to carry out high-temperature stress experiments. The fishes were first cultured for 3 weeks at 18°C in 12-L tanks to acclimate to the environment, and they were fed a commercial diet twice a day throughout the study. For temperature-stress experiments, five temperatures in a 2°C gradient, 18, 20, 22, 24 and 26°C, were used. Fishes were randomly sampled after 24 h in water at the target temperatures of 18, 20, 22 and 24°C. Our pilot study found that the fishes started to die when the water temperature rose to 26°C. Thus, we took samples at 4, 24, 48 and 72 h as the water temperature rose from 24 to 26°C. In summary, a total of eight sampling points (temperature–time) were used (Fig. 1), 18°C-24h, 20°C-24h, 22°C-24h, 24°C-24h, 26°C-4h, 26°C-24h, 26°C-48h and 26°C-72h.
2.2 Sample collection and RNA extraction
For each individual, six tissues including liver, blood, heart, brain, gill, and skin, were collected for total RNA extraction. At each sampling point, five fishes were randomly chosen for tissue dissection. The tissues were stored in liquid nitrogen until total RNA was extracted using TRIzol reagent (Thermo Fisher, CA, USA) following the recommended protocol17. After treating with DNaseI (Fermentas, MD, USA) to eliminate genomic DNA, the RNA quality was assessed using a 1.0% agarose gel and a Nano Drop™ 8000 spectrophotometer (Thermo Fisher, CA, USA), and the RNA quantity was measured using a Qubit3 kit (Thermo Fisher, CA, USA).
2.3 Reference gene selection and primer design
Using the transcriptome published by Tong18, the transcript sequences of 10 genes, 28S rRNA, GAPDH, ARBPR, 18S rRNA, β-actin, RPS29, RPL13, RPL19, Saha and α-tubulin, were determined for qRT-PCR primer design. The primers were first designed using Primer3 https://primer3plus.com/ (Table 1) and then aligned to the taimen transcriptome using a BLAST search to confirm the specificity19. Primers that matched non-target genes with less than 3 mismatched bases were eliminated. The primers were synthesised by GENEWIZ Co. (Suzhou, China).
Table 1
Primer sequences and qPCR efficiencies of the 10 candidate taimen reference genes
Gene
(Full Name)
|
Accession number
|
Sequence (5'→3')
|
Product
Size (bp)
|
PCR
Efficiency (%)
|
18S rRNA
(18S ribosomal RNA)
|
HAGJ01000001
|
F: CGTTCTTAGTTGGTGGAGCG
|
123
|
89.3
|
R: AACGCCACTTGTCCCTCTAA
|
Saha
(S-adenosyl-L-homocysteine hydrolase)
|
HAGJ01162991
|
F: TGGAGGGATGGCTGAACATT
|
131
|
102.6
|
R:AGAGCACTGGAGGAAACACA
|
GAPDH (Glyceraldehyde-3-phosphate dehydrogenase)
|
HAGJ01029436
|
F: GTCTTCTGGGTAGCGGTGTA
|
128
|
93.3
|
R: ACCATCGTCAGCAATGCATC
|
RPL13
(ribosomal protein L13)
|
HAGJ01015081
|
F: GGCCATCTTGAGTTCCTCCT
|
161
|
100.9
|
R: GCACCATTGGCATCTCTGTT
|
ARBP
(attachment region binding protein )
|
HAGJ01017568
|
F: GGGCTTTGTCTTCACCAAGG
|
145
|
100.0
|
R: CTTCTCAGGACCAAGCCCAG
|
28S RNA
(28S ribosomal RNA)
|
HAGJ01147709
|
F: GTCCTTCTGATCGAGGCTCA
|
125
|
100.3
|
R: GGAGTTTACCACCCGCTTTG
|
β-actin
(actin, beta)
|
HAGJ01168534
|
F: TCTACGAAGGCTACGCTCTG
|
155
|
109.4
|
R: CAGCTTCTCCTTGATGTCGC
|
RPL19
(ribosomal protein L19)
|
HAGJ01117629
|
F:ACACGGGCATAGGTAAGAGG
|
124
|
106.9
|
R: TCGATTTTCTTGGCCTCCCT
|
RPS29
(ribosomal protein S29)
|
HAGJ01023957
|
F:TGGGACATCAGAGCCTCTAC
|
122
|
110.5
|
R: CTGGCGGCACATGTTGAG
|
α-tubulin
(alpha-Tubulin)
|
HAGJ01091547
|
F:CGAGCCATACATCACACACG
|
137
|
107.8
|
R: TGCAATTGGGTGTTGATCCA
|
Hsp60
|
HAGJ01009947
|
F: GACATCATCAGACGAGCCCT
R: ACGTACTCTCCTTCCATGGC
|
137
|
105.4
|
Hsp70
|
HAGJ01160617
|
F: CCGCCTGGTTAGTCACTTTG
R: AGTGTTCTCTTGGCCCTCTC
|
117
|
110.3
|
2.4 Quantitative real-time PCR
The cDNA was synthesised using a RevertAid™ First-Strand cDNA Synthesis Kit (Fermentas, MD, USA) with oligo(dT)18 as the primer and stored at − 20°C. The qRT-PCR was carried out in a 10-µL volume, which included 1 µL cDNA (50 ng/µL), 5 µL 2× Luna universal SYBR qPCR Master Mix (New England Biolabs, MA, USA), 0.5 µL each primer (10 µM) and 3 µL H2O. The amplification program was as follows: 95°C for 15 s, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. A melting curve was performed from 60 to 95°C. Triple technological replicates were used for each sample. To calculate the qRT-PCR efficiency of each gene, 10-, 100- and 1,000-fold diluted cDNA samples were used to create a standard curve, and a linear regression model was built with the log10 (concentration) as the independent variable. The corresponding qRT-PCR efficiencies (E) were calculated using the following equation: E = [10(− 1/slope) − 1] × 10020,21.
2.5 Stability analysis of candidate reference genes
The relative expression levels of the candidate reference genes were calculated using Ct values and amplicon mean amplification efficiencies20. To assess the stability levels of the 10 reference genes, four programs, geNorm13, NormFinder14, BestKeeper15 and RefFinder16, were used. The Ct values of the 10 candidate genes were analysed from 48 experiments having two experimental factors. The first factor was the six tissues (blood, heart, brain, gill, skin and liver), and the second factor was the eight sets of different heat-stress conditions, 18°C-24h, 20°C-24h, 22°C-24h, 24°C-24h, 26°C-4h, 26°C-24h, 26°C-48h and 26°C-96h.