The isolation of total RNA and RT–qPCR were performed as previously reported28 (link). Briefly, total RNA was isolated using RNAiso Plus (Takara, Dalian, China) according to the manufacturer’s instructions. Subsequently, 1st-strand cDNA was transcribed using a HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). To measure vRNA copies or the transcriptional expression of cytokines, RT–qPCR assays were performed using 2× Taq SYBR Green qPCR Premix (Innovagene, Changsha, China) with a CFX Connect Real-Time PCR Detect System (Bio–Rad) following the manufacturer’s protocols. All of the related primers for RT–qPCR are presented in Table S1 .
Cfx connect real time pcr detect system
Manufactured by Bio-Rad
Sourced in China
The CFX Connect Real-Time PCR Detection System is a compact and flexible instrument designed for real-time PCR analysis. It provides high-quality data and reliable performance for a wide range of applications.
Automatically generated - may contain errors
4 protocols using cfx connect real time pcr detect system
1
RNA Extraction and RT-qPCR for Cytokine Expression
2
RNA Isolation and RT-qPCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total RNA was isolated using RNAiso Plus regent (Takara, Dalian, China) per manufacturer’s instruction. To measure vRNA level in tissues or the transcriptional expression of cytokines in spleens, reverse transcription quantitative PCR (RT-qPCR) assays were performed using 2×Taq SYBRGreen qPCR Premix (Innovagene, Changsha, China) in a CFX Connect Real-Time PCR Detect System (Bio-rad) following the manufacturer’s protocols. Primers used in present study are presented in Table S1
3
Quantitative RT-PCR Analysis of Transferred OT1 Cells
4
Real-Time qPCR Analysis of Intestinal Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples of the duodenal and jejunal mucosal were homogenized with an appropriate amount of TransZol. Total RNA was extracted from the duodenal and jejunal mucosal according to the instructions of TransZol Up Plus RNA kit. Then, the RNA was reverse transcribed into cDNA according to the instructions of the cDNA Synthesis SuperMix kit. Finally, real-time qPCR was performed on a CFX Connect Real-Time PCR Detect System (Bio-Rad, California, USA) according to the instructions of PerfectStartTM Green qPCR SuperMix kit. The above kits were purchased from Beijing TransGen Biotech Co., Ltd. (Beijing, China). The primer sequences used in this study, as shown in Table 4 , were designed and synthesized by Shanghai Generay Biotechnology Co., Ltd. (Shanghai, China). β-actin acts as the normalization of targeted genes. The 2−ΔΔCt method was used to calculate the relative mRNA expression.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!