Sybr green pcr kit

B7-H6 mRNA expression was assessed in Huh7, HepG2, HepG2.215, LO2, K562, and HeLa cells by quantitative real-time PCR. The details were as follows: Total RNA was extracted from cell samples using TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions. Subsequently, Total RNA (1μg) was used for cDNA synthesis using a QuantiTect Reverse transcription Kit (Qiagen, Hilden, German). Real-time PCR was performed using a SYBR green PCR kit (TOYOBO, Japan) in an ABI 7500 Sequence Detection System (Applied Biosystems, Sunnyvale, CA). The sequences of the primers used to amplify B7-H6 were 5'-TCACCAAGAGGCATTCCGACCT-3' (sense) and 5'-ACCACCTCACATCGGTACTCTC-3' (anti-sense). The housekeeping gene GAPDH was used as an internal control.

The total RNA from HepG2 cells and mouse primary hepatocytes was isolated using RNAiso Plus (Takara, Japan). Reverse transcription was carried out with RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA). The primers (Wuhan Qinke Innovation Biotechnology, China) are described in Table 1. The annealing temperature for SIK1 amplification was 52.5°C, while the internal control gene β-actin was amplified at 50°C. The PCR products were separated by agarose gel electrophoresis and visualized on a JS-680B-Imaging System (Shanghai Peiqing Science and Technology, China). SIK1 mRNA amounts in each sample were normalized to β-actin expression. Analysis was performed by densitometry with ImageJ (National Institutes of Health, USA).
qRT-PCR was carried out with an SYBR Green PCR kit (Toyobo, Japan) on an AB7500 RT-PCR (Applied Biosystems, USA), using the housekeeping gene GAPDH for normalization. The primers used for quantitating human and mouse genes by qRT-PCR are listed in Table 1.

Total RNA was isolated using TRIzol reagent and treated with DNase I to remove genomic DNA contamination as previously described27 (link). The synthesis of first-strand cDNA and RT-qPCR were performed by use of Maxima^® First Strand cDNA Synthesis kit (Fermentas, St Leon-Rot, Germany) and SYBR^® Green PCR kit (Toyobo, Osaka, Japan), respectively according to the manufacturer’s instructions. PCR primers were designed using Primer 3 software (http://frodo.wi.mit.edu/as) (Table S1, see Supporting Information), and glyceraldehyde-3-phosphate dehydrogenase (gapdh), whose cycle threshold (Ct) values were not changed upon TDCIPP exposure in this study (Figure S1), was used as an internal reference. The mRNA levels were expressed as fold change using the 2^-ΔΔCt method. There were 3 replicated tanks for each concentration, and three fish from each tank were used and thus totally 9 fish were analyzed in each treatment.

RNA isolation and RT-PCR were performed according to the manufacturer’s instructions. Total RNA was extracted with Trizol reagents following the manufacturer’s instructions (Invitrogen). MRNA levels of primary miRNAs, SOX4, and CUL4B were assayed by SYBR Green PCR kit (Toyobo, Osaka, Japan) and mature miRNA expression levels were quantitated using MicroRNA Assay Kit (Takara, Otsu, Japan). GAPDH and U6 were used as an endogenous control for mRNA and miRNA, respectively. Primers used were described in Supplementary Table 2.

Total RNA and cDNA were extracted and prepared as previously described [14 (link)]. Quantitative PCR was performed using the SYBR Green PCR Kit (Toyobo, Osaka, Japan) on a CFX-96 Real-Time PCR system (Bio-Rad, Hercules, CA, USA). The primer pairs RT-catB-F/RT-catB-R and RT-actA-F/RT-actA-R (Table S8) were used to quantify the catB and actA genes, respectively. The relative mRNA levels were normalized to that of the reference gene actA using the 2^−ΔΔCt method of relative quantification [14 (link),19 (link)]. The experiment was repeated thrice. The mean values ± SD of the three independent experiments were calculated based on one-way analysis of variance with Dunnett’s post hoc test, which were used to identify the statistical differences (* p < 0.05; ** p < 0.01; and *** p < 0.001).