Sybr green pro taq hs premix 2

Total RNA was extracted from cells using TRIzol reagent (Takara, Kyoto, Japan).
Reverse transcription was implemented using Evo M-MLV RT Kit with gDNA Clean for qPCR (Accurate Biology, #AG11728, Changsha, China). For RT-qPCR, 2x SYBR Green Pro Taq HS Premix II (Accurate Biology, #AG11701) was used and performed using the QuantStudio 5 real-time-PCR instruments (ThermoFisher, USA). Data were obtained from at least three independent experiments. Primer sequences used in this study were listed in Supplementary Table 3.

Total RNA from the hindgut was extracted using TransZol Up Plus RNA Kit (Beijing TransGen Biotech Co., Ltd., Beijing, China) and the integrity of total RNA was validated by agarose gel electrophoresis; the purity and concentration of total RNA were analyzed spectrophotometrically (A260:280 nm). Then, reverse-transcription of RNA to cDNA was performed using PrimeScript^TM RT Reagent Kit (Takara Bio Inc., Tokyo, Japan) according to the instruction. Based on the sequences in Gen Bank, specific primers were designed using Primer 5, as shown in Table 2. Real-time quantitative PCR was executed on a quantitative thermal cycler (Light Cycler 480, Roche Diagnostics, Switzerland). The 10 μL reaction volume for RT-qPCR contains 5 μL 2X SYBR^® Green Pro Taq HS PremixII (Accurate Biotechnology (Hunan) Co., Ltd., Hunan, China), 3.8 L RNase-free water, 1 μL of cDNA and 0.1 μL of two primers (5 μM each). The thermal profile for real-time quantitative PCR was 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 60 °C for 30 s, and one cycle of 95 °C for 5 s, 60 °C for 60 s, then 50 °C for 30 s. β-actin was considered as the reference gene. Gene expression results were calculated according to the 2^−ΔΔCT method [22 (link)].

RT-qPCR was carried out on a 384-well board with the total reaction volume is 10 µL, involving 5 µL of 2X SYBR® Green Pro Taq HS Premix II (Accurate Biology, Hunan, China), 3.2 µL of RNase Free dH₂O, 0.8 µL of every primer and 1 µL for cDNA sample. The PCR reaction criteria were set by a thermal program: 95°C for 30 min, forty circles of 95°C for 5 s, next 60°C for 34 s. Every sample was performed in triplicate. We designed the primers of the reference gene (β-actin) and the target genes based on the published grouper genes sequence (Table 2). The threshold cIRCLE value of every sample was collected since processing. The related gene expression was estimated by the 2^−ΔΔCt approach [48 (link)].

Total RNA was isolated from mouse liver tissues with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and was converted to cDNA with the Evo M-MLV RT Master Mix (Accurate Biotechnology, Changsha, China). A quantitative real-time PCR (qRT-PCR) was performed on Bio-Rad CFX 96 (Bio-Rad Laboratories, Hercules, CA, USA) with SYBR^® Green Pro Taq HS Premix II (Accurate Biotechnology). The relative mRNA expression levels were assessed using the 2^−ΔΔCq method and normalized by the Actb (used as a control gene). The primers were designed from IDT DNA Technology (Coralville, IA, USA) and synthesized by Shenggong Bioengineering Co., Ltd. (Shanghai, China). The primer sequences used for the PCR are shown in Table S1.

To extract total RNA, the rat renal tissues (50 mg) or cultured HK-2 cells were homogenized in 500 μL TRIzol^® reagent (Invitrogen, Waltham, MA, USA). The total RNA pellet was dissolved in diethylpyrocarbonate (DEPC) water. The quality and quantity of total RNA were detected using ultraviolet absorption (optical density, 260 nm/280 nm) with a NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). An equal amount of total RNA (500 ng) was reverse transcribed to cDNA using Evo M-MLV RT Master Mix (Accurate Biotechnology, Hunan, China). Quantitative real-time PCR (qRT-PCR) was performed on Bio-Rad CFX 96 (Bio-Rad, Hercules, CA, USA) with SYBR^® Green Pro Taq HS Premix II (Accurate Biotechnology). The relative mRNA expression levels were assessed using the 2^−ΔΔCq method and normalized using gene GAPDH (used as an internal control gene). The primers were designed from IDT DNA Technology (Coralville, IA, USA) and synthesized by Sangon Biotech, Co., Ltd. (Shanghai, China). The primer sequences used for PCR are shown in Table S1.

The experimental cells were lysed by Trizol Reagent kit and total RNA was extracted. The Evo M-MLV RT kit (Accurate Biotechnology, Changsha, Hunan, China) was used for reverse transcription of RNA into cDNA. The qRT-PCR was performed by a kit containing 2 $\times$ SYBR^® Green Pro Taq HS Premix II (Accurate Biotechnology, Changsha, Hunan, China). The amplification conditions were as follows: an initial denaturation at 95°C for 3 min, followed by 40 cycles at 95°C for 15 s, 57°C for 15 s, and 72°C for 20 s. Gene expression was quantified relative to β-actin expression using the 2^−ΔΔCt method. The sequence of β-actin forward primer: 5′-ATA​AAT​GGA​GCA​CCT​CAA​CCT-3′, reverse primer: 5′-TCT​CCT​TCT​GCG​AGC​GTC​CT-3'. The sequence of Omentin-1 forward primer: 5′-TGG​CCA​GAG​GGA​ATT​TAC​TGC-3′, reverse primer: 5' -CAC​CGA​TGC​AGT​GAT​GTT​CAG- 3'. The sequence of GAS6 forward primer: 5′-CGA​TTA​ACC​CTC​GCC​TGG​AT-3′, reverse primer: 5′-GCG​ATG​TTC​GGG​TGT​AGT​TG-3'. All experiments were performed in triplicate.