Talent qpcr premix with sybr green 1

For validation, the 1st-strand cDNA of brain, gill, liver and muscle tissue of five fish under 27 °C, 23 °C, 19 °C, 15 °C and 13 °C stress was synthesized via PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, RR047) based on the manufacture’s protocol. The gene-specific primers of differently expressed genes HSP70, CRIBP, TULP4, WWP2, PPARD, CUL9, USP4 and reference gene βACTIN were designed according to Primer3web (http://primer3.ut.ee/) (Supplementary Table 5). The βACTIN gene sequence identification and stability evaluation was conducted previously (data unpublished). Quantitative real-time PCR (qPCR) was conducted using Talent qPCR PreMix with SYBR GREEN I (Tiangen, FP209) on an ABI FastOnePlus machine (Applied Biosystems). A two-step program method was performed as following: 95 °C 3 min; 40 cycles of 95 °C for 5 s and 60 °C for 30 s. After the program melting curve analysis was done. The data was analyzed using 2^-△△Ct relative quantification method [79 (link)] with βACTIN as inner control. All the experiment was conducted in triplicates. qPCR data was analyzed by StepOne software (version 2.3) with P < 0.01. The significance was calculated by one-way ANOVA and post-hoc test with S-N-K and Tukey methods.

With the extracted RNA of the goldfish (CN, BK, and WH) as templates, the first strand of cDNA was synthesized via PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, RR047) based on the manufacture’s protocol. The qPCR primers of differently expressed genes coro1c, sept2b, cldnda, nect2, myh, and col12a1 and reference gene β2m/gapdh were designed according to Primer3web (http://primer3.ut.ee/ (accessed on 29 July 2022)) (Supplementary Table S4). The reference genes were selected and evaluated according to the transcriptomic data. qPCR was conducted using Talent qPCRPreMix with SYBR GREEN I (Tiangen, Beijing, China, FP209) on an FastOne machine (Applied Biosystems, Thermofisher, Waltham, MA, USA). The program was performed as following: 95 °C 3 min; 40 cycles of 95 °C for 5 s; and 60 °C for 30 min. After the program melting curve analysis was conducted, the data were analyzed using the 2-^△△Ctrelative quantification method with B2M/GAPDH as an inner control in triplicates. The significance was calculated by one-way ANOVA and a post hoc test with the SNK and Tukey methods, p < 0.01.