Real-time quantitative PCR analyses was performed using a CFX96 Real-Time System (Bio-Rad Laboratories, Hercules, CA) using the SYBR Green PCR Core Reagents (Beijing TransGen Biotech, Beijing, China). The reaction mixture (10 μL) contained 1 μL template cDNA, 5 μL SYBR Green I, 3.4 μL DEPC water, 0.2 μL ROX Reference Dye (50×), and 0.2 μL each of forward and reverse primers. Thermal conditions for PCR were as follows: pre-denaturation 95°C for 10 s, followed by 45 cycles of 95°C for 10 s, 54.7°C for 30 s, and 75°C for 15 s. Each sample was run in triplicate for analysis. At the end of the PCR cycles, melting curve analysis was performed to validate the specific generation of the expected PCR product. The relative mRNA expression ratio of target genes were calculated using the 2−ΔΔCt method (Livak and Schmittgen, 2001 (link)).
Sybr green pcr core reagents
Manufactured by Transgene
SYBR Green PCR Core Reagents are a set of reagents designed for use in polymerase chain reaction (PCR) experiments. The core function of these reagents is to facilitate the amplification and detection of target DNA sequences through the use of the SYBR Green I dye, which binds to double-stranded DNA.
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Lab products found in correlation
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Transscript first strand complementary dna, by Transgene (1 mentions)
Cdna synthesis supermix, by Transgene (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
2 protocols using sybr green pcr core reagents
1
Real-Time qPCR Analysis Protocol
2
Quantification of Immune Chemokine Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Monocyte chemoattractant protein 1 (MCP-1), CCL-3, and CCL-5 mRNA expressions were analyzed by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Total RNA was extracted from splenic lymphocytes using Trizol Reagent (Invitrogen) and total RNA was reverse-transcribed using TransScript First-Strand complementary DNA (cDNA) Synthesis SuperMix (TransGen Biotech), according to the manufacturer’s instructions. The PCR primers were designed according to the NCBI database (Table 1 ) and PCR was performed with a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) using SYBR Green PCR Core Reagents (TransGen Biotech). Amplification of β-actin mRNA was used as an endogenous control. There were 10 replicates per group and each sample was assayed in triplicate and a mean value was calculated. Data were analyzed according to the 2–ΔΔCt method and results were expressed as relative mRNA levels. Each group was assayed 18 times (three replicates per group and each sample assayed in sextuplicate).
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