Total RNA was isolated from the cells using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer’s manuals. Total RNA (1 μg) was reverse-transcribed using a 2× cDNA synthesis premix kit (BioFACT, Seoul, Korea). Real-time reverse transcription PCR was performed using SYBR Green qPCR Master Mix (BioFACT) and a real-time PCR system (Bio-Rad Laboratories, Hercules, CA, USA). The primer sequences utilized are shown in Table 1 . The relative amounts of mRNA were calculated based on the cycle threshold values using β-actin as a control. All experiments were performed in triplicate and the values were averaged.
Sybr green qpcr master mix
Manufactured by Biofact
Sourced in United States
SYBR Green qPCR Master Mix is a ready-to-use solution for real-time quantitative PCR (qPCR) analysis. It contains SYBR Green I dye, which binds to double-stranded DNA and emits a fluorescent signal upon binding, allowing for real-time detection and quantification of PCR amplicons.
Automatically generated - may contain errors
Lab products found in correlation
Real time pcr system, by Bio-Rad (2 mentions)
Cdna synthesis kit, by Biofact (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Tri reagent, by Molecular Research Center (1 mentions)
Rna extraction kit, by GeneAll (1 mentions)
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
5 protocols using sybr green qpcr master mix
1
Total RNA Extraction and qRT-PCR Analysis
2
Quantitative RT-PCR Protocol for Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted using RNA extraction kit (GeneAll, Seoul, Korea) according to the manufacturer’s protocols. Total RNA was reversed-transcribed to cDNA with cDNA synthesis Kit (BioFACT, Seoul, Korea). Real-time RT-PCR was performed using SYBR Green qPCR Master Mix (BioFACT, Seoul, Korea) and a Real-time PCR system (Bio-Rad, Hercules, CA, USA). Primer sequences are shown in Table 1 . Relative amounts of mRNAs were calculated based on the threshold cycle number using β-actin as an endogenous control. All experiments were performed in triplicate and values were averaged.
3
Real-Time PCR Analysis of Inflammatory Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Real-time PCR was performed using StepOnePlus Real-Time PCR System (Applied
Biosystems, Foster City, CA, USA) with 10 ng cDNA, SYBR Green qPCR Master Mix
(Cat# DQ385-40h, Biofact, Daejeon, Korea), and 1 pM primer (Table 1 ). Levels of TLR3,
tumor necrosis factor (TNF)
receptor-associated factor 3 (TRAF3),
Toll-like receptor adaptor molecule 1(TICAM1), nuclear factor-κB1(NF-κB1), TNFα, and
cyclooxygenase2 (COX2) mRNA were measured
[21 (link)]. mRNA fold-change was normalized
to β-actin mRNA using
2−ΔΔCt method [22 (link)].
Biosystems, Foster City, CA, USA) with 10 ng cDNA, SYBR Green qPCR Master Mix
(Cat# DQ385-40h, Biofact, Daejeon, Korea), and 1 pM primer (
tumor necrosis factor (TNF)
receptor-associated factor 3 (TRAF3),
Toll-like receptor adaptor molecule 1(TICAM1), nuclear factor-κB1(NF-κB1), TNFα, and
cyclooxygenase2 (COX2) mRNA were measured
[21 (link)]. mRNA fold-change was normalized
to β-actin mRNA using
2−ΔΔCt method [22 (link)].
4
RNA Isolation and qPCR Analysis of Liver Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total RNA was isolated from liver tissue homogenates using TRIzol reagent (Takara, Shiga, Japan), according to the manufacturer’s instructions. Complementary DNA was synthesized from RNA using a cDNA synthesis kit (BioFact, Daejeon, Korea). Relative gene expression was determined using SYBR Green qPCR Master Mix (BioFact) on a QuantStudio 3 real-time PCR system (Life Technologies, Carlsbad, CA, USA). The fold change in gene expression compared to LFD-fed mice was calculated using the comparative threshold cycle (Ct) method, which was normalized to mouse Gapdh. The primers used in this study are listed in Additional file 1 Supplementary Table 1.
5
Quantification of Immune Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Real-time PCR was performed using StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with 10 ng cDNA, SYBR Green qPCR Master Mix (Cat# DQ385-40h, Biofact, Daejeon, Korea), and 1 pM primer (Table 2). Levels of TLR3, TNF receptor-associated factor 3 (TRAF3), Toll-Like Receptor Adaptor Molecule 1 (TICAM1), nuclear factor-κB1 (NF-κB1), tumor necrosis factor α (TNFα), and cyclooxygenase2 (COX2) mRNA were measured [21] . mRNA fold-change was normalized to β-actin mRNA using 2 -ΔΔCt method [22] .
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!