Total RNA was isolated using Agilent Total RNA Isolation Mini kit to the manufacturer’s instructions. Total 500 ng of RNA was reverse transcribed into cDNA using ReverTra Ace qPCR RT Master Mix kit (FSQ-201; TOYOBO). The relative mRNA expression levels were determined using real-time quantitative PCR by THUNDERBRID SYBR qPCR Mix (QPS-201; TOYOBO). Relative mRNA expression levels were determined by the ddCt method. Beta-actin was used as reference internal standard. The qPCR primers were picked up from published papers (Plotkin et al., 2014 (link); Luo et al., 2017 (link); Jerónimo-Santos et al., 2015 (link); Kursan et al., 2017 (link)), the sequence described as follow: TrkB FL: 5′-AAGGAGCCTTCGGGAAAGTT-3′ (forward), 5′-GAAAGTCCTTGCGTGCATTG-3′ (reverse), TrkB T1: 5′-CACTGGATGGGTAGCTGAGATA-3′ (forward), 5′-TGCAGACATCCTCGGAGATT-3′ (reverse), KCC2: 5′-TGCCCAGAAGTCTATCCCTAC-3′ (forward), 5′-CACCAAGTTGCCATTCACAG-3′ (reverse), PARP1: 5′-TGGTTTCAAGTCCCTTGTCC-3′ (forward), 5′-TGCTGTCTATGGAGCTGTGG-3′ (reverse), KIF4: 5′-GAGCACACTAAAATGTCAGGAGG-3′ (forward), 5′-CTGTTTGCTGGCTACTTGGAG-3′ (reverse), Act: 5′-AGCCATGTACGTAGCCATCC-3′ (forward), 5′-CTCTCAGCTGTGGTGGTGAA-3′ (reverse).
Thunderbrid sybr qpcr mix
Manufactured by Toyobo
Sourced in United States, Japan
THUNDERBRID SYBR qPCR Mix is a ready-to-use solution for real-time quantitative PCR (qPCR) analysis using SYBR Green detection. It contains all the necessary components for efficient and sensitive qPCR amplification, including a proprietary DNA polymerase, SYBR Green I dye, and optimized buffer system.
Automatically generated - may contain errors
Lab products found in correlation
Revertra ace qpcr rt master mix kit, by Toyobo (1 mentions)
Total rna isolation mini kit, by Agilent Technologies (1 mentions)
Genespringgx 7, by Agilent Technologies (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Revertra ace qpcr rt master mix, by Toyobo (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rneasy lipid tissue mini kit, by Qiagen (1 mentions)
Tri reagent, by Takara Bio (1 mentions)
Thermal cycler dice real time system, by Takara Bio (1 mentions)
3 protocols using thunderbrid sybr qpcr mix
1
Quantitative Real-Time PCR Assay for Gene Expression
2
Quantitative Real-Time PCR and Microarray Analysis
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Total RNA was isolated from the cells or tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and an RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany). Total RNAs were used to synthesize the cDNA using a ReverTra Ace®qPCR RT Master Mix (TOYOBO, Osaka, Japan) with random primers. The cDNA was used to amplify selected genes with THUNDERBRID® SYBR® qPCR Mix (TOYOBO) and primers using the Applied Biosystems QuantStudio 3 Real-Time PCR (Applied Biosystems, Foster city, CA, USA). Expression levels and dene specific primer sets were described previously [28 (link),29 (link),30 (link)].
For the microarray analysis, total RNAs from C3H10T1/2 adipocytes treated with 10 μM of sesamol or tBHQ for 24 h were isolated and cleaned using an RNeasy Mini Kit (Qiagen, Venlo, The Netherlands). To assess the reproducibility of the RNA amplification, three samples each of DMSO, tBHQ, and sesamol RNA were amplified independently. The cDNA preparation and hybridization to Affymetrix Mouse Genome Arrays of 430 version 2.0 were performed by Macrogen (Seoul, Korea). Data were analyzed using the GeneSpring GX 7.3 software (Agilent Technologies, Santa Clara, CA, USA).
For the microarray analysis, total RNAs from C3H10T1/2 adipocytes treated with 10 μM of sesamol or tBHQ for 24 h were isolated and cleaned using an RNeasy Mini Kit (Qiagen, Venlo, The Netherlands). To assess the reproducibility of the RNA amplification, three samples each of DMSO, tBHQ, and sesamol RNA were amplified independently. The cDNA preparation and hybridization to Affymetrix Mouse Genome Arrays of 430 version 2.0 were performed by Macrogen (Seoul, Korea). Data were analyzed using the GeneSpring GX 7.3 software (Agilent Technologies, Santa Clara, CA, USA).
3
Duodenal Immune Gene Expression
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The total RNA was extracted from the duodenum using Tri Reagent™ (TaKaRa Bio Inc., Shiga, Japan) following standard protocol. The quality and concentration were determined, and the first strand cDNA was synthesized using 2.5 μg of total RNA by PrimeScript™ cDNA kit (TaKaRa, Bio Inc., Shiga, Japan). Expression of selected immune response genes was performed by qRT-PCR (Thermal Cycler Dice Real Time System, TaKaRa Bio Inc., Shiga, Japan). Reaction mixture (14 μL) included 5 μL of cDNA as a template, 7 μL of THUNDERBRID® SYBR® qPCR mix (Toyobo Co., Ltd., Osaka, Japan) and 1 μL of gene specific forward and reverse primers (10 pmol/μL). Relevant primer sequences are included in Supplementary Table S2 . Data were normalized using GAPDH (housekeeping gene) and expression fold was calculated by 2−ΔΔCT method [45 (link)].
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