Sybr green

Total RNA was extracted from the rat fetal heart tissues using Trizol reagent (Life Technologies) according to the manufactures instructions. Reverse transcription (RT-PCR) was performed using the Access RT-PCR System (Promega). Quantitative PCR was performed with SYBR Green (TaKaRa) used as a double-stranded DNA-specific fluorescent dye Reactions were conducted with 1 μL RT-PCR cDNA, 0.5 μL each of forward and reverse primers (10 μmol/L), 8μL water and 10 μL SYBR Green. The samples were run by the StepOne real-time PCR machine (ABI, USA).β-actin was used to normalize the data. Primer pairs for β-actin, Smad3, Dhfr, Sumo3, Pdp1 Mt-atp6 were showed in S1 Table). qRT-PCR was performed in triplicate.

According to the manufacturer’s instructions, total RNA was extracted from fresh whole blood utilizing TriPure isolation reagent (Roche, Germany). cDNA was synthesized employing the RNJia PB Kit (ROJETechnologies, Iran). SYBR Green-based RT-qPCR was employed by SYBR Green (TaKaRa, Otsu, Japan), according to the manufacturer’s instructions. The relative 2 standard curves real-time PCR was performed on the cDNA samples using Q-6000 machine (Qiagen, Germany). The GAPDH gene and U6 miRNA were utilized as the housekeeping genes to normalize the mRNA and miRNAs expression levels respectively, as well as to control the error between samples [13 (link),14 (link)]. The list of the designed primers and probes for determining the expression levels of SIRT1, FZD6, THBS4, CPNE3 genes and miRNAs including miR-451a [15 (link),16 (link)] and miR-142 [17 (link),18 (link)] are indicated in Table 2.

Total RNA was isolated from cells or tissues using Trizol (Takara). The RNA concentration and purity were determined spectrophotometrically. And cDNA was synthesized using approximately 1μg of total RNA and the reverse transcriptase M-MLV (Takara Bio, Shiga, Japan). Quantitative real-time PCR (RT-PCR) was carried out using 2× SYBR green (Takara Bio, Shiga, Japan) with specific primers for ZIPs (1 (link)–14 (link)), YM1, ARG1, IL6 and β-Actin (Table 1) in a C1000 thermal cycler (BIO-RAD CFX96, Hercules, CA, USA). β-Actin was used for normalization. The relative mRNA expression was calculated using the 2^−ΔCT method or 2^-ΔΔCT method.

Three fish were randomly selected from each tank and dissected. The liver was immersed in RNAlater (Ambion, USA) and then stored at -80°C for the mRNA expression assays. Total RNA of the samples were extracted using the commercial kit (Beijing All-Style Gold Biotechnology Co., Ltd.). The RNA quality was assessed with agarose gel electrophoresis and ultramicroscopic spectrophotometer (NanoDrop-1000, Wilmington, USA). The cDNA was obtained using the Prime Script™ RT reagent kit (Takara, Japan). Real-time fluorescent quantitative PCR (LightCycler480) was performed using SYBR® Green Master Mix (Takara, Japan). The 10 μL reaction system consisted of 5 μL 2× SYBR Green (Takara, Japan), 1 μL cDNA, 0.5 μL primers, and 3 μL sterile double-distilled water. The processes were as follows: 95°C denaturation step for 30 s, 40 amplification cycles “denaturation at 95°C for 5 s, annealing at 60°C for 30 s,” followed by melt curve analysis and cooling to 4°C. The relative mRNA expression levels of the target genes were calculated according to equation 2^-ΔΔCt using β-actin as the housekeeping gene [25 (link)]. The primer sequences are presented in Table 2.

Total RNAs from the gingiva, liver, and adipose tissue were extracted using an RNeasy Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Total RNA was quantified by measuring the absorbance at 260–280 nm. Subsequently, aliquots of RNA were reverse transcribed to complementary DNA (cDNA) using a Primescript RT Master Mix Kit (Takara Bio, Shiga, Japan). Quantitative real-time PCR analysis was performed using an Applied Biosystems 7500 Fast Real-time PCR System (Life Technology, Carlsbad, CA, USA) in accordance with the manufacturer’s protocol. Briefly, the reactions were carried in a 10-μl mixture of 2× SYBR Green (Takara Bio, Shiga, Japan), each primed at 100 nM and 30 ng of reverse-transcribed RNA. The PCR program consisted of a 30-s incubation at 95°C, followed by 40 cycles at 95°C for 5 s and 60°C for 34 s. A dissociation curve analysis was then performed to confirm specificity. Each gene was tested in triplicate. The relative level of messenger RNA (mRNA) for each target mRNA was determined using the 2^−ΔΔCt method. The relative quantities were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA and represented in fold changes relative to those from control group mice. The sequences of the primers are provided in Table 1.