The mRNA expression of ER stress-related genes (grp78, grp94, xbp-1, pdi-3) and apoptosis-related genes (bcl-1, bcl-2, bax, chop) in liver tissues was determined by real-time PCR according to previous reports (Mo et al., 2009 (link); Mo et al., 2012 (link)). In brief, total RNA was extracted from liver tissues with Trizol reagent (Qiagen, Germany). The cDNA was synthesized from 0.5 µg of total RNA with a cDNA synthesis kit (Takara, Dalian, China). All primers (Table 1 ) were purchased from Haofeng Biotechnology (China). The PCR was performed in duplicate on a CFX-Touch 96 (Bio-Rad, USA) by using SYBR Green PCR Master Mix (Takara, Dalian, China). The reaction parameters for real-time PCR were one cycle at 95°C for 4 min, followed by 40 cycles, with each cycle at 95°C for 5 s, 60°C for 20 s, and 65°C for 30 s. The relative mRNA expression level of the gene was normalized to the level of GAPDH in the same sample.
Cfx touch 96
Manufactured by Bio-Rad
Sourced in United States
The CFX-Touch 96 is a real-time PCR detection system manufactured by Bio-Rad. It is designed to perform quantitative and qualitative gene expression analysis, genotyping, and other nucleic acid detection applications. The system features a touch-screen interface, a 96-well sample block, and compatibility with a variety of sample types and reagents.
Automatically generated - may contain errors
Lab products found in correlation
Cdna synthesis kit, by Takara Bio (1 mentions)
Trizol reagent, by Qiagen (1 mentions)
Sybr green pcr master mix, by Takara Bio (1 mentions)
Cdna reverse transcription kit, by Vazyme (1 mentions)
Sybr green supermix, by Vazyme (1 mentions)
Sybr green 1 master mix, by Thermo Fisher Scientific (1 mentions)
3 protocols using cfx touch 96
1
Quantification of ER Stress and Apoptosis Genes
2
Quantitative Gene Expression Analysis
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Total RNA was extracted with TRIzol reagent and a complementary DNA (cDNA) reverse transcription kit (R223-01; Vazyme, Nanjing, China) was used to prepare cDNA. Gene expression analysis was conducted using SYBR Green Supermix (Vazyme, R223-01) in CFX connect light cycler (CFX-Touch 96; Bio-Rad, Hercules, CA, USA), according to the manufacturer’s instructions. A total of 1,000 ng RNA of each sample was subjected to RT-qPCR experiments and samples were tested in triplicate. The primer sequences are listed in Table S1 .
3
Quantitative PCR Analysis of CSF1 Breakpoint
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Real‐time quantitative PCR amplification of cDNA was performed to verify and analyze the CSF1 breakpoint identified by transcriptome sequencing. Based on the RNA sequence findings, primers were designed around the putative breakpoint in the CSF1 gene. Primers with positive PCR products are listed in Table 1 . For the CSF1‐S100A10, we used primers published by Panagopoulos et al.23 The PCR amplifications were performed using the Thermo Fisher Applied Biosystems SYBR green real‐time PCR protocol, using 12.5 μl of SYBR® Green I master mix (Life Technologies, Carlsbad), 1.5 μl of the forward and reverse primers each, and 1 μl of cDNA (1:10 diluted). The PCR was run on CFX touch 96 (BIO‐RAD, Hercules) with the cycling profile of initial denaturation for 3 min at 95°C followed by 40 cycles of 15 s at 95°C, 20 s at 55°C, and 60 s at 72°C.
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