The total RNA was isolated from cells using the RNeasy kit (Qiagen, Valencia, CA, USA). The cDNA was synthesized by the iScriptTM cDNA Synthesis system (Bio-Rad, Hercules, CA, USA). The real-time RT-PCR was performed by using the SensiFAST SYBR qPCR mix (Bioline, London, UK) according to the manufacturer’s protocol. The relative values of gene expression were normalized to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primer sequences are provided in Table 1 .
Sensifast sybr qpcr mix
Manufactured by Meridian Bioscience
Sourced in United Kingdom
SensiFAST SYBR qPCR mix is a ready-to-use solution for quantitative PCR (qPCR) analysis. It contains all the necessary components for efficient and sensitive real-time PCR amplification of DNA targets, including a hot-start DNA polymerase, dNTPs, and a SYBR Green I fluorescent dye.
Automatically generated - may contain errors
8 protocols using sensifast sybr qpcr mix
1
Quantitative Gene Expression Analysis by RT-PCR
2
RNA Extraction and cDNA Synthesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolation of total RNA using the Direct-zol™ RNA kit (Zymo Research, Orange, CA, USA) and synthesis of cDNA using the iScript™ cDNA Synthesis kit (Bio-Rad, Hercules, CA, USA) followed the manufacturer’s protocol. The transcripts were amplified using the SensiFAST SYBR qPCR mix (Bioline, London, UK) [31 (link)]. Table 1 shows the primers used for amplification.
3
RNA Isolation and Real-time PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total RNA were isolated from the cells using a Direct-zol™ RNA kit (Zymo Research, Orange, CA, USA). The cDNA was synthesized using an iScript™ cDNA Synthesis System (Bio-Rad). Real-time PCR was performed using a SensiFAST SYBR qPCR mix (Bioline, London, UK), according to the manufacturer’s protocol. The gene expression values were normalized relative to 18S expression values. The primer sequences used in these experiments are listed in Table 1 .
4
RNA Isolation and Real-Time RT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total RNA was isolated from cells using the Direct-zol™ RNA kit (Zymo Research, Orange, CA, USA). The cDNA was synthesized by the iScript™ cDNA Synthesis system (Bio-Rad, Hercules, CA, USA). The reak-time RT-PCR was accomplished by using the SensiFAST SYBR qPCR mix (Bioline, London, UK) according to the manufacturer’s protocol. The relative values of gene expression were normalized to GAPDH. The primer sequences are provided in Table 1 .
5
Quantification of Inflammatory Cytokines in Colon Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from the colon lysate using the Direct-zol RNA kit (Zymo Research, Orange, CA, USA). The cDNA was synthesized using the iScript cDNA Synthesis System (Bio-Rad, Hercules, CA, USA). Quantitative RT-PCR was performed using the SensiFAST SYBR qPCR mix (Bioline, London, UK) according to the manufacturer’s protocol. The primer sequences of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and 18S ribosomal RNA (18 s) used in this study are reported in Table 2 . The values of gene expression were normalized to those of 18 s.
List of primers used for real time RT-PCR
| Symbol | Full name | Primer sequence (5’-3’) | |
|---|---|---|---|
| Forward | Reverse | ||
| Tnf-α | Tumor necrosis factor-α | GGCCTCTCTACCTTGTTGCC | CAGCCTGGTCACCAAATCAG |
| Il-6 | Interleukin-6 | TTGCCTTCTTGGGACTGATG | CCACGATTTCCCAGAGAACA |
| Il-1β | Interleukin-1β | TTCACCATGGAATCCGTGTC | GTCTTGGCCGAGGACTAAGG |
| 18S | 18S ribosomal RNA | CAGCCACCCGAGATTGAGCA | TAGTAGCGACGGGCGGTGTG |
6
Quantifying Gene Expression Using qPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total RNA was harvested from cells using the Direct-zol RNA kit (Zymo Research, Orange, CA, USA). The cDNA was synthesized by the iScript cDNA Synthesis system (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocols. Amplification of transcripts was performed by using the SensiFAST SYBR qPCR mix (Bioline, London, UK). The optimal number of PCR cycles was determined for each primer set to ensure a linear range of amplification. The relative values of gene expression were normalized to 18 s. The primer sequences are provided in Table 3 .
7
RNA Extraction and RT-PCR Analysis of Liver Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total RNA was isolated from liver tissue and cells using the RNeasy kit (Qiagen, Valencia, CA, USA). Then, the cDNA was synthesized by using the iScript™ cDNA Synthesis system (Bio-Rad, Hercules, CA, USA). The real-time RT-PCR was performed by using the SensiFAST SYBR qPCR mix (Bioline, London, UK) according to the manufacturer’s protocol. The relative values of gene expression were normalized to 18S ribosomal RNA. The primer sequences and full gene names are provided in Table 3 .
8
Quantitative RT-PCR Analysis of Hepatic Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantitative RT-PCR was determined as reported previously [16 (link)]. Total RNA was isolated from the liver lysate using the Direct-zol RNA kit (Zymo Research, Orange, CA, USA). cDNA was synthesized with a iScript cDNA Synthesis system (Bio-Rad, Hercules, CA, USA). Quantitative RT-PCR was performed using the SensiFAST SYBR qPCR mix (Bioline, London, UK) according to the manufacturer’s protocol. The values of gene expression were normalized to those of GAPDH. Primer sequences are provided in Table 1 .
Primers used for quantitative RT-PCR
| Genes | Primer Sequences | |
|---|---|---|
| TNF-α | F: GGCCTCTCTACCTTGTTGCC | R: CAGCCTGGTCACCAAATCAG |
| IL-6 | F: TTGCCTTCTTGGGACTGATG | R: CCACGATTTCCCAGAGAACA |
| IL-1β | F: TTCACCATGGAATCCGTGTC | R: GTCTTGGCCGAGGACTAAGG |
| α-SMA | F: GCACCCAGCATGAAGATCAAG | R: TCTGCTGGAAGGTAGACAGCGAAG |
| TGFβ1 | F: GCCCTGGATACCAACTATTGC | R: TGTTGGACAGCTGCTCCACCT |
| Col1a1 | F: ACCTGTGTGTTCCCTACTCA | R: GACTGTTGCCTTCGCCTCTG |
| GAPDH | F: GTTGTCTCCTGCGACTTCA | R: GGTGGTCCAGGGTTTCTTA |
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!