Sybr green pro taq hs

Cells were cultured in 12-well plates, and each mandarin fish received 100 μL virus or an equal volume of DMEM by intraperitoneal injection. The RNA samples were reverse-transcribed to cDNA using a PrimeScript RT Reagent Kit (Takara, China) after the total RNA from transfected or infected cells or fish tissue was extracted using a total RNA extraction kit (Promega, China). RT-qPCR assays were conducted using SYBR Green Pro Taq HS (Accurate Biology, China) with a Roche LightCycler® 480 System, while S. chuatsi β-actin or GAPDH was used as the reference gene. The reaction programs were as follows: 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 15 s. The primers used are listed in Supplementary Data 3.

To extract RNA from HUVECs, aortic tissues and CDNVs were isolated and homogenized in TRIzol reagent (Life Technologies, USA). RNA was reverse transcribed to cDNA using the Evo M-MLV Mix Kit (AG11728, ACCURATE BIOTECHNOLOGY (HUNAN) Co., LTD, Changsha, China). Quantitative real-time PCR (qPCR) analysis was performed using a LightCycler480 (Roche, USA). The primer sequences are listed in Additional file 1: Table S1. The expression of miRNAs in tissues and HUVECs were normalized to U6 snRNA, and the levels of VCAM-1, ICAM-1, and CXCL12 were normalized to GAPDH with SYBR® Green Pro Taq HS (AG11702, Accurate Biotechnology (Hunan) CO., LTD, ChangSha, China). For miRNAs in CDNVs detection, the synthetic miRNA Caenorhabditis elegans miR-39 (cel-miR-39; 10 fmol/µL; Sequence: 5ʹ-UCACCGGGUGUAAAUCAGCUUG-3′; AG, Accurate Biotechnology (Hunan) CO., LTD, ChangSha, China) was added to the isolated RNAs and was used as an exogenous control. The expression levels of the tested genes were determined and calculated by 2−∆∆Ct. Each data point contained at least three biological duplicates and is represented as the mean ± standard deviation.

Total RNAs from platelets were reverse transcribed to cDNA using Evo M-MLV RT Kit (Accurate Biotechnology (Hunan) Co., Ltd., Changsha, China). qRT-PCR for selected LncRNAs were performed on cDNAs using SYBR Green Pro Taq HS (Accurate Biotechnology (Hunan) Co., Ltd., Changsha, China). GAPDH was used as the internal control. The relative gene expression of lncRNAs was calculated by comparing the threshold cycle (C_T). Each sample was tested in triplicate.

RT-qPCR was performed using SYBR Green Pro Taq HS (Accurate Biology, Changshang, China). RT-qPCR primers were designed using TBtools. The relative expression levels were calculated using the e 2^−∆∆Ct method. The RT-qPCR procedure was followed according to a previously established protocol [90 (link)]. GAPDH was used as the internal reference gene.

Total RNA was extracted from the colon and hypothalamus samples using RNA isolation kit (R0027, Beyotime, Beijing, China) and then reverse transcribed to cDNA with Reverse Transcription Kit (K1622, Thermo Scientific, Waltham, MA, USA). Gene expression was quantified by qPCR using SYBR Green Pro Taq HS (AG11701, Accurate Biology, Changsha, Hunan, China) in an iQ5 PCR cycler (Bio-Rad, Hercules, CA, USA) with specific primers. All the results were analyzed using the −ΔΔCt method and normalized to the reference gene Actb. The primer sequences are listed in Table 1.

The details of total RNA extraction using 1 ml of Trizol (TRI Reagent solution, Invitrogen, Carlsbad, CA, USA), cDNA preparation using an Evo M-MLV reagent Kit with gDNA Eraser (Accurate Biotechnology (Hunan) Co., Ltd), and qPCR assays using SYBR® Green Pro Taq HS (Accurate Biotechnology (Hunan) Co., Ltd) are provided in our previous study [2 (link), 27 ]. Primers used in this study included in Supplementary 5: fatty acid synthase (fas), srebp1, pparγ, adipose triglyceride lipase (atgl), pparα, acyl-CoA oxidase 1 (aco), delta-6 fatty acyl desaturase (fad6), elongase of very long-chain fatty acid 4 (elovl4), elovl8, fatty acid binding protein (fabp), uncoupling protein 2 (ucp2), and liver X receptor alpha (lxr). β-actin and 18s rRNA were used as reference genes to normalize the genes expression. The results of gene expression were calculated using the 2^−ΔΔCT method [28 , 29 ].

Total RNA was isolated using AG RNAex Pro Reagent (Accurate Biology, Changsha, China) and transcribed into cDNA using the Evo M-MLV Kit (Accurate Biology, Changsha, China). Quantitative real-time PCR (qRT-PCR) analysis was carried out using SYBR® Green Pro Taq HS (Accurate Biology, Changsha, China) under the following conditions: 95 ℃ for 30 s; 40 cycles of 95 ℃ for 5 s and 60 ℃ for 30 s, with final melting-curve analysis. The primer sequences used are listed in Table 1. Relative gene expression was calculated by normalizing the housekeeping gene β-actin in mouse mammary gland tissue or GAPDH in primary GMECs using the comparative 2^-∆∆Ct method.Table 1

Primers sequences for qRT-PCR

Gene	Sequence (5′ → 3′)	Length, bp
Goat Nrf2	F: CCAACTACTCCCAGGTAGCCC R: AGCAGTGGCAACCTGAACG	227
Goat HO-1	F: CAAGCGCTATGTTCAGCGAC R: GCTTGAACTTGGTGGCACTG	206
Goat NQO1	F: ACTGTGTCGGACCTGTATGC R: CAGAGAGTACATGGAGCCGC	363
Goat GCLM	F: AATCTTGCCTCCTGCTGTGTGATG R: GATGCTCTCCTGAAGTGCTTCTTGG	138
Goat GCLC	F: CATTTGCAAAGGTGGCAACGC R: CTGCTTGTAGTCGGGATGCT	301
Goat GAPDH	F: ACCTGCCAAGTATGATGAG R: AGTGTCGCTGTTGAAGTC	118
Mouse TNF-α	F: ACGGCATGGATCTCAAAGAC R: GTGGGTGAGGAGCACGTAGT	116
Mouse IL-1β	F: GCTGCTTCCAAACCTTTGAC R: AGCTTCTCCACAGCCACAAT	121
Mouse IL-6	F: CCGGAGAGGAGACTTCACAG R: CAGAATTGCCATTGCACAAC	134
Mouse β-actin	F: GTCAGGTCATCACTATCGGCAAT R: AGAGGTCTTTACGGATGTCAACGT	147