Total RNA was isolated from MAC-T cells using TRIzol (Invitrogen) according to the manufacturer's protocol. Reverse transcription was performed using a quantitative real-time PCR Master Mix with gDNA Remover (Toyobo, Osaka, Japan) according to the manufacturer's instructions. Quantitative realtime PCR was performed using the SYBR Premix Ex TaqTM ll (TaKaRa Biotechnology, Kusatsu, Japan). Gene expression of XBP1s, ATF4, ATF6A, and CHOP were assessed. Relative transcript expression was calculated via the 2 -ΔΔCt method and represented as values relative to control. The GAPDH and VPS4A genes were used as references. Primer sequences were used according to Yonekura et al. (2018) (link). The primer sequence of GAPDH was AATGGAAAGGCCATCA (forward) and GTGGTTCACGCCCATC (reverse). The primer sequence of VPS4A was CAAAGCCAAGGAGAG-CATTC (forward) and ATGTTGGGCTTCTCCAT-CAC (reverse). Sensitivity of the reaction and amplification of contaminating products, such as extension of self-annealed primers, were evaluated by amplifying serial cDNA dilutions. Data analysis was performed according to the manufacturer's instructions.
Sybr premix ex taqtm ll
Manufactured by Takara Bio
Sourced in Japan
SYBR Premix Ex TaqTM II is a real-time PCR reagent used for quantitative gene expression analysis. It contains SYBR Green I dye, Taq DNA polymerase, and necessary components for efficient PCR amplification.
Automatically generated - may contain errors
3 protocols using sybr premix ex taqtm ll
1
Quantitative Real-Time PCR Analysis of UPR Genes
2
Quantification of mRNA Expression
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Total RNA was extracted from the HC11 cells using TRIzol reagent (Life Technologies) following the manufacturer’s protocol. A qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan) was utilized for reverse transcription. Quantitative real-time PCR was carried out using the SYBR Premix Ex TaqTM ll (TaKaRa Biotechnology, Kusatsu, Japan), to quantify the relative expression level of mRNA. Relative mRNA expression was estimated by the double delta delta Ct method and represented as the relative values to the control. GAPDH was used as the housekeeping gene. The primers used for the quantitative PCR are shown in Table 1 . The reaction sensitivity and amplification of the contaminating products were examined by amplifying serial dilutions of cDNA.
3
Quantitative Real-Time PCR Analysis of Gene Expression
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Total RNA was isolated from mammary glands or MAC-T cells using TRIzol (Invitrogen) according to the manufacture's protocol. For quantitative real-time (qRT)-PCR, cDNA were synthesized from total RNA using a qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan).
Quantitative real-time PCR was performed in a Thermal Cycler Dice Real Time System (TaKaRa Bio Inc., Shiga, Japan) by using SYBR Premix Ex TaqTM ll (TaKaRa Bio Inc.). The primer sequences are shown in Table 1. Relative expression was quantified using the standard curve method and data were normalized to β-actin gene (ACTB) expression. All qRT-PCR was performed on Thermal Cycler Dice Real Time System (TaKaRa Bio Inc.), based on a standard curve method. The sensitivity of the reaction and amplification of contaminating products, such as the extension of selfannealed primers, were evaluated by amplifying serial dilutions of cDNA. All data analysis was performed as recommended by the manufacturer.
Quantitative real-time PCR was performed in a Thermal Cycler Dice Real Time System (TaKaRa Bio Inc., Shiga, Japan) by using SYBR Premix Ex TaqTM ll (TaKaRa Bio Inc.). The primer sequences are shown in Table 1. Relative expression was quantified using the standard curve method and data were normalized to β-actin gene (ACTB) expression. All qRT-PCR was performed on Thermal Cycler Dice Real Time System (TaKaRa Bio Inc.), based on a standard curve method. The sensitivity of the reaction and amplification of contaminating products, such as the extension of selfannealed primers, were evaluated by amplifying serial dilutions of cDNA. All data analysis was performed as recommended by the manufacturer.
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