Total RNA was extracted using an SV Total RNA Isolation Kit (Promega, USA) and reversed with a ReverTra Ace qPCR RT Kit (TOYOBO, Japan) according to the manufacturer's protocol as described previously (43 (link)). qPCR was performed in an Applied Biosystems QuantStudio 5 Real Time Detection System (Thermofisher, USA) with the primers listed in Table 1 . Each assay was carried out in triplicate under the following cycling conditions: 95°C for 1 min for activation, followed by 40 cycles at 95°C for 15 s, 60°C for 15 s, and 72°C for 45 s. The expression level of viral, host IFN, and ceramide synthesis-related genes was normalized to β-actin and calculated using the 2−ΔΔCT method. The data were represented as the mean ± standard deviation (SD).
Quantstudio 5 real time detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The QuantStudio 5 Real Time Detection System is a thermal cycler device designed for real-time PCR analysis. It is capable of performing quantitative, qualitative, and multiplex PCR experiments. The system provides detection using fluorescence-based technologies and can accommodate a variety of sample plate formats.
Automatically generated - may contain errors
Lab products found in correlation
Revertra ace qpcr rt kit, by Toyobo (3 mentions)
Sv total rna isolation kit, by Promega (2 mentions)
Sybr green realtime pcr master mix, by Toyobo (2 mentions)
Bulge loop mirna qrt pcr kit, by RiboBio (1 mentions)
Cell total rna isolation kit, by Foregene (1 mentions)
Sybr green real time pcr mix, by Toyobo (1 mentions)
5 protocols using quantstudio 5 real time detection system
1
Quantification of Viral and Host Transcripts
2
Quantification of miR-124 Expression
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MiR-124 level was determined using Bulge-Loop™ miRNA qRT-PCR kit (RiboBio, China), and the U6 was used as the reference genes. qPCR was performed with SYBR Green Real-Time PCR Master Mix (Toyobo) at Applied Biosystems QuantStudio 5 Real Time Detection System (Thermo Fisher, USA). The reaction system was 10 µl including 5 µl of SYBR qPCR mix, 0.3 µl of forward and reverse bulge-loop miRNA primer, 3.4 µl of PCR-grade water, and 1 µl of diluted miR-124-specific cDNA. The reaction condition was 95°C for 5 min, followed by 40 cycles of 94°C for 5 s, 56°C for 10 s, and 72°C for 15 s. The expression of the genes was normalized to reference gene and calculated with the 2−ΔΔCt method.
3
Quantitative Analysis of E. coioides DUSP5
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The expression patterns of E. coioides DUSP5 were assessed using quantitative real-time PCR amplification (qPCR), and the cDNA was used as a template. The primers are listed in Table 1 . The primers DUSP5- RT-F and DUSP5-RT-R were designed for qPCR, and β-actin was used as the reference gene. qPCR was performed with SYBR Green Real-Time PCR Master Mix (Toyobo) using an Applied Biosystems QuantStudio 5 Real Time Detection System (Thermo Fisher, Waltham, MA, USA). The conditions were 1 cycle of 95 °C for 1 min followed by 40 cycles of 95 °C, 15 s; 60 °C, 15 s; 72 °C, 45 s. The expression of the target gene was normalized to the reference gene and calculated with the 2−ΔΔCt method.
4
RNA Extraction and qRT-PCR Analysis
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A Cell Total RNA Isolation Kit (FORE GENE, Chengdu, China) was used to extract total RNA, and a ReverTra Ace qPCR RT Kit (TOYOBO, Osaka, Japan) was used for the reverse transcription of RNA, as described previously [26 (link)]. An Applied Biosystems QuantStudio 5 Real Time Detection System (Thermo Fisher, Waltham, MA, USA) with the cyclic conditions described previously was used for qRT-PCR analysis [22 (link)]. The primers used in the experiment are listed in Table 2 . The relative expression ratio of the selected gene vs. β-actin (reference gene) was calculated using the 2−∆∆Ct method.
5
RNA Extraction and qPCR Analysis
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Total RNA was extracted using the SV Total RNA Isolation Kit (Promega) and cDNA was synthesized using the ReverTra Ace qPCR RT Kit (Toyobo) following the manufacturer's protocol. qPCR was performed on an Applied Biosystems QuantStudio 5 Real Time Detection System (Thermofisher, USA) using 2 × SYBR Green Real-time PCR Mix (Toyobo) with the following PCR conditions: 95 °C for 1 min for activation, followed by 40 cycles at 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 45 s. The qPCR primers are listed in Table 1 . Relative mRNA levels were calculated with the 2−ΔΔCt method with β-actin as the internal control. Data are presented as mean ± standard deviation (SD) of three independent experiments.
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