Total RNA was extracted from cell lines using the TRIzol™ Reagent (Invitrogen) and was reverse-transcribed into single-stranded complementary DNA using the PrimeScriptTM RT Reagent Kit (Takara Bio Inc.). Subsequent quantitative PCR amplification was performed with SYBRTM Premix Ex Taq™ (TaKaRa Bio Inc.) on an Applied Biosystems 7500 real-time detection system (Applied Biosystems, Carlsbad, CA, USA). Primers for IL-12, IL-10, tumor necrosis factor α (TNF-α), transforming growth factor β1 (TGF-β1), CCL5, and CCR5 cDNAs were designed by Sangon Biotech and are listed in Table S1
7500 real time detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 7500 real-time detection system is a laboratory instrument designed for accurate and reliable quantitative real-time PCR analysis. It is capable of detecting and quantifying a wide range of target DNA or RNA sequences with high sensitivity and specificity.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Revertaid first strand cdna synthesis kit, by Takara Bio (1 mentions)
Sybr premix ex tag kit, by Takara Bio (1 mentions)
Genelute mammalian genomic dna miniprep kit, by Merck Group (1 mentions)
Tb green advantage qpcr premix, by Takara Bio (1 mentions)
3 protocols using 7500 real time detection system
1
Quantitative PCR Analysis of Cytokine Expression
2
Quantifying Piezo1 Expression in Primary Chondrocytes
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To verify the expression of key genes from the forementioned analysis, mice primary chondrocytes were isolated and cultured [27 (link)]. Yoda 1 was used to treat chondrocytes to activate the expression of Piezo1. The total RNA was extracted from the cells using TRIzol reagent (Invitrogen). cDNA was synthesized using a RevertAid First Strand cDNA Synthesis Kit (TaKaRa). qRT-PCR was conducted to amplify cDNA using an SYBR Premix Ex Tag Kit (TaKaRa) and a 7500 Real-time detection system (Applied Biosystems, Waltham, MA, USA). The primers used in this work were designed by Sangon Biotech Co., Ltd. (Shanghai, China), and the sequences of these primers are listed in Table 1 . The relative expression of each gene was normalized to Gapdh and presented in a heatmap after normalization (log10 transformation).
3
ASFV DNA Quantification via qPCR
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Tissue samples were ground and lysed before viral nucleic acid extraction. ASFV genomic DNA was extracted from the tissue using GenElute Mammalian Genomic DNA Miniprep Kits (Sigma–Aldrich, USA). ASFV genomic DNA was quantified with TB Green Advantage qPCR Premix (Takara) on an Applied Biosystems 7500 Real Time Detection System (Roche, Germany) to determine the gene expression levels using the β‐actin gene promoter as a control. Each assay was performed in triplicate, and the level was calculated by the 2−∆∆ct method. The quantitative real‐time PCR (qPCR) results were consistent with the sequencing results.
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