Total RNA was extracted by using TRIzol solution (TaKaRa, 9109) according to the manufacturer’s instructions. Then, the RNA was reverse transcribed into cDNA on a PCR amplifier by using a PrimeScript RT Reagent Kit (TaKaRa, RR037A). Then, qPCR was performed by using TB Green Premix Ex Taq II (TaKaRa, RR820A) according to the manufacturer’s directions, and gene expression was measured by using a real-time fluorescence quantitative PCR system (Applied Biosystems, 7500). GAPDH served as the reference gene, and the relative gene expression was determined with the 2−ΔΔCt method. The relevant primers are listed in Supplementary Table 1 .
Real time fluorescence quantitative pcr system
Manufactured by Thermo Fisher Scientific
The real-time fluorescence quantitative PCR system is an advanced instrument used for the amplification and detection of nucleic acid sequences in real-time. It utilizes fluorescence-based technology to quantify the amount of target DNA or RNA present in a sample during the amplification process.
Automatically generated - may contain errors
Lab products found in correlation
Tb green premix ex taq 2, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Trizol solution, by Takara Bio (1 mentions)
Sybr premix ex taq 2, by Thermo Fisher Scientific (1 mentions)
Evo m mlv rt master mix, by Accurate Biology (1 mentions)
Sybr green pro taq hs premix, by Accurate Biology (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
3 protocols using real time fluorescence quantitative pcr system
1
Quantitative RT-qPCR Gene Expression Analysis
2
RNA Extraction and qPCR Analysis Protocol
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Total RNA was isolated from cells or lung tissues using TRIzol reagent, RNA was reverse transcribed into cDNA according to the PrimeScript RT Reverse Transcription Kit, and mRNA expression was quantified using the Applied Biosystems Real-Time Fluorescence Quantitative PCR System and the SYBR premix Ex Taq II. Quantification results were calculated using the 2-ΔΔCT method for comparison with β-actin as the reference mRNA (Table 1 ).
Primer sequences of the target genes
| Target | Sequence (5′–3′) | Orientation | |
|---|---|---|---|
TLR4 TLR4 | 5’-AAACCACTCCACTCCCTCAG-3’ 5’-CTTCTGGTCCTTGACCCACT-3 | Forward Reverse | |
IL-10 IL-10 | 5’-CTGAGAACAGCTGCATCCAC-3’ 5’-AAAGTCCTCCAGCAGAGACC-3’ | Forward Reverse | |
SOD1 SOD1 | 5’- ATCAAGAGAGGCACGTTGGA-3’ 5’- GGGCGATCACAGAATCTTCG − 3’ | Forward Reverse | |
CAT CAT | 5’- ACATGGTCTGGGACTTCTGG-3’ 5’- CATGTGCCTGTGTCCATCTG-3’ | Forward Reverse | |
Nqo1 Nqo1 | 5’- GCTTACACATACGCTGCCAT-3’ 5’- GCCACAGAAATGCAAAGTGC-3’ | Forward Reverse | |
COX2 COX2 | 5’- AAAGCCTTGCTGTTCCAACC-3’ 5’- TTGGAGTGGGCTTCAGGAAT-3’ | Forward Reverse | |
Nos2 Nos2 | 5’- GGGTCAGAGCTACCATCCTC-3’ 5’- CGTCCATGCAGAGAACCTTG-3’ | Forward Reverse | |
β-actin β-actin | 5’-GGTCACCAGGGCTGCTTT-3’ 5’-ACTGTGCCGTTGACCTTGC-3’ | Forward Reverse | |
3
RNA Extraction and qPCR Analysis
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The RNA was extracted using TRIzol™ Reagent (Thermo Fisher, 15596-026) according to the manufacturer’s instructions. The concentrations of the extracted RNA were measured and equal amount of RNA was reversely transcribed into cDNA using Evo M-MLV RT Master Mix (Accurate Biology, AG11706). The qPCR was performed on cDNA by using SYBR® Green Pro Taq HS Premix (Accurate Biology, AG11701) on a Real-time fluorescence quantitative PCR system (Applied Biosystems, ABI7500). The relative RNA levels were analyzed in 2−ΔΔCt method with GAPDH as the reference gene. The primers used in this study are showed in Table S2 .
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