Quantitative real-time PCR (qRT-PCR) was performed on a MyiQTM2 Two-Color Real-Time PCR Detection System (Bio-Rad) using SYBR PrimeScript Ex Taq II kit (TaKaRa) according to the manufacturer's instructions. β–actin was served as an internal reference gene. The prime pairs used for qRT-PCR detection of poP2RX7, β-actin, IL-1β and IL-6 are F5/R4, F6/R5, F7/R6 and F8/R7, respectively (Table 1 ). qRT-PCR was performed using an initial denaturation at 95°C for 30 s, 40 cycles at 95°C for 5 s, 60°C for 30 s followed by dissociation curve analyses (55°C to 95°C: increment 0.5°C for 5 s). Relative expression levels of the target genes in experimental group versus those in control group were determined with the comparative 2−△△Ct quantification method [25] (link). Data are presented as means ± standard deviation from triplicate experiments. Statistical analysis was performed with Student's t-test for the comparison between two groups. Multiple group comparison was conducted by one-way ANOVA followed by Duncan's analysis. Differences were considered significant at p<0.05.
Sybr primescript ex taq 2 kit
Manufactured by Takara Bio
The SYBR PrimeScript Ex Taq II kit is a reagent designed for real-time PCR applications. It contains the necessary components for performing quantitative and qualitative analysis of DNA samples, including a DNA polymerase enzyme and fluorescent dye for detection.
Automatically generated - may contain errors
2 protocols using sybr primescript ex taq 2 kit
1
Quantitative Real-Time PCR Analysis of Gene Expression
2
Evaluating eATP Impact on Immune Response
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To test the effects of eATP on cytokine and immune-related gene expression,
Japanese flounder HKM and PBL cells were treated with 200 or 1000 μM ATP (cell
culture grade, Sigma-Aldrich) for the indicated times, and eATP-induced gene
expression changes in IL-1β, IL-6, IL-11, TNF-α, G-CSF, IFN, Mx, and NF-κ B p65
subunit (p65) were quantified by quantitative real-time PCR (qRT-PCR). For this
aim, one microliter of cDNAs from each source was amplified in a MyiQ™ Two-Color
Real-Time PCR Detection System (Bio-Rad) with corresponding primer pairs (Table 1 ) in a total
volume of 25 μl using SYBR PrimeScript Ex Taq™ II kit (TaKaRa) under the
following conditions: initial denaturation at 95°C for 30 s, 40 cycles at 95°C
for 5 s, 60°C for 30 s, followed by dissociation curve analysis (55°C to 95°C:
increment 0.5°C for 5 s). β–Actin was used as an internal
reference gene. Agarose gel electrophoresis analyses were performed at the end
of each qRT-PCR to validate specificity of amplification. The identities of all
the qRT-PCR products were further verified by DNA sequencing. The results are
expressed as fold changes in the target gene normalized to the reference gene
and as relative to expression in untreated controls. Data are presented as the
means ± SEM from triplicate experiments.
Japanese flounder HKM and PBL cells were treated with 200 or 1000 μM ATP (cell
culture grade, Sigma-Aldrich) for the indicated times, and eATP-induced gene
expression changes in IL-1β, IL-6, IL-11, TNF-α, G-CSF, IFN, Mx, and NF-κ B p65
subunit (p65) were quantified by quantitative real-time PCR (qRT-PCR). For this
aim, one microliter of cDNAs from each source was amplified in a MyiQ™ Two-Color
Real-Time PCR Detection System (Bio-Rad) with corresponding primer pairs (
volume of 25 μl using SYBR PrimeScript Ex Taq™ II kit (TaKaRa) under the
following conditions: initial denaturation at 95°C for 30 s, 40 cycles at 95°C
for 5 s, 60°C for 30 s, followed by dissociation curve analysis (55°C to 95°C:
increment 0.5°C for 5 s). β–Actin was used as an internal
reference gene. Agarose gel electrophoresis analyses were performed at the end
of each qRT-PCR to validate specificity of amplification. The identities of all
the qRT-PCR products were further verified by DNA sequencing. The results are
expressed as fold changes in the target gene normalized to the reference gene
and as relative to expression in untreated controls. Data are presented as the
means ± SEM from triplicate experiments.
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