Cell mRNA was extracted 6 h post-infection (p.i) according to the manufacturer’s instructions (Qiagen) and cDNA generated using the Quantitect cDNA Synthesis Kit (Qiagen) as previously described [32 ]. Incubation time was chosen based on maximal expression of interferon (IFN)-β by DCs following S. suis infection [32 ]. Real-time qPCR was performed on the CFX-96 Touch Rapid Thermal Cycler System (Bio-Rad) using 250 nM of primers (Integrated DNA technologies) and the SsoFast Evagreen Supermix Kit (Bio-Rad). Cycling conditions were 3 min of polymerase activation at 98°C, followed by 40 cycles at 98°C for 2 sec and 57°C for 5 sec. Melting curves were generated after each run to confirm presence of a single PCR product. The primer sequences used in this study are shown in Table 2 and were verified to have reaction efficiencies between 90% and 110%. The reference genes Atp5b and Gapdh, determined to be the most stably expressed using the algorithm geNorm, were used to normalize data. Fold changes in gene expression were calculated using the quantification cycle threshold (Cq) method using the CFX software manager v.3.0 (Bio-Rad). Samples from mock-infected cells served as calibrators. IFN-β was measured by RT-qPCR in order to compare with published results [32 ].
Cfx 96 touch rapid thermal cycler system
Manufactured by Bio-Rad
Sourced in United States
The CFX-96 Touch Rapid Thermal Cycler System is a real-time PCR instrument that enables fast and accurate quantification of nucleic acids. It features a 96-well format and provides precise temperature control for efficient amplification of DNA and RNA samples.
Automatically generated - may contain errors
4 protocols using cfx 96 touch rapid thermal cycler system
1
Measuring Interferon-beta Expression in S. suis Infected Cells
2
Real-time qPCR Gene Expression Analysis
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Cell mRNA was extracted according to the manufacturer’s instructions (TRIzol) and cDNA generated using the Quantitect cDNA Synthesis Kit (Qiagen, Mississauga, ON, Canada). Real-time qPCR was performed on the CFX-96 Touch Rapid Thermal Cycler System (Bio-Rad, Hercules, CA, USA) using 250 nM of primers (Integrated DNA technologies, Coralville, IA, USA) and the SsoFast Evagreen Supermix Kit (Bio-Rad). The cycling conditions were 3 min of polymerase activation at 98°C, followed by 40 cycles at 98°C for 2 s and 57°C for 5 s. Melting curves were generated after each run to confirm the presence of a single PCR product. The sequences of primers used in this study are shown in Table S1 in Supplementary Material and were verified to have reaction efficiencies between 90% and 110%. The reference genes Atp5b and Gapdh, determined to be the most stably expressed using the algorithm geNorm, were used to normalize data. Fold changes in gene expression were calculated using the quantification cycle threshold (Cq) method using the CFX software manager v.3.0 (Bio-Rad). Samples from mock-infected cells served as calibrators.
3
Quantitative RT-qPCR Analysis of Gene Expression
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Cell mRNA was extracted according to the manufacturer’s instructions (TRIzol). RNA purity was assessed by spectrophotometric quantification and integrity verified by electrophoresis on denaturating agarose gel. cDNA was generated using the Quantitect cDNA Synthesis Kit (Qiagen, Mississauga, ON, Canada) with 500 ng of RNA pretreated with DNase. Real-time qPCR was performed on the CFX-96 Touch Rapid Thermal Cycler System (Bio-Rad) using 250 nM of primers (Integrated DNA technologies), the SsoFast Evagreen Supermix Kit (Bio-Rad) and 20 ng of cDNA. No template controls were included and all samples were run in triplicate. The cycling conditions were 3 min of polymerase activation at 98 °C, followed by 40 cycles at 98 °C for 2 s and 57 °C for 5 s. Melting curves were generated after each run to confirm the presence of a single PCR product. The sequences of primers used in this study are shown in Additional file 1 and were verified to have reaction efficiencies between 90 and 110%. The reference genes Atp5b and Gapdh, determined to be the most stably expressed using the algorithm geNorm, were used to normalize data. Fold changes in gene expression were calculated using the quantification cycle threshold (Cq) method using the CFX software manager v.3.0 (Bio-Rad). Samples from mock-infected cells served as calibrators.
4
Quantitative Real-Time PCR Analysis of Gene Expression
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Cell mRNA was extracted according to the manufacturer’s instructions (TRIzol). RNA purity was assessed by spectrophotometric quantification and integrity verified by electrophoresis on denaturating agarose gel. cDNA was generated using the Quantitect cDNA Synthesis Kit (Qiagen, Mississauga, ON, Canada) with 500 ng of RNA pretreated with DNase. Real-time polymerase chain reaction (qPCR) was performed on the CFX-96 Touch Rapid Thermal Cycler System (Bio-Rad), using 250 nM of primers (Integrated DNA technologies), the SsoFast Evagreen Supermix Kit (Bio-Rad) and 20 ng of cDNA. No template controls were included and all samples were run in triplicate. The cycling conditions were 3 min of polymerase activation at 98 °C, followed by 40 cycles at 98 °C for 2 s and 57 °C for 5 s. Melting curves were generated after each run to confirm the presence of a single PCR product. The sequences of primers used in this study are shown in Table 2 and were verified to have reaction efficiencies between 90% and 110%. The reference genes Atp5b and Gapdh, determined to be the most stably expressed using the algorithm geNorm, were used to normalize data. Fold changes in gene expression were calculated using the quantification cycle threshold (Cq) method using the CFX software manager v.3.0 (Bio-Rad). Samples from mock-infected cells served as calibrators.
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