For qRT-PCR experiments, total RNA from colon epithelia was extracted using RNAiso Plus by following the manufacturer's instructions (Takara, Japan). Total RNA (500 ng) was converted to cDNA, and PCR reactions were performed in triplicate using a SYBR Green PCR LightCycler® (Roche Diagnostics, Indianapolis, Indiana, USA) in a 96-well format over 45 cycles with denaturation at 95°C for 10 s and annealing at 58°C for 20 s. The primers for mouse β-actin, A3AR, TNF-a, and IL-1β were purchased from Sangon Biotech (Shanghai, China). The sequences of these primers were listed in Table 1 . Data were normalized versus β-actin for mouse A3AR, TNF-a, and IL-1β by using a 2−ΔΔCT method. Data were expressed as the fold change in mRNA transcript levels relative to that of the normal control.
Mouse β actin
Manufactured by Sangon
Sourced in China
Mouse β-actin is a cytoskeletal protein found in eukaryotic cells. It is involved in various cellular processes, including cell motility, structure, and integrity. This protein is commonly used as a reference or control in various laboratory techniques, such as Western blotting, immunohistochemistry, and gene expression analysis.
Automatically generated - may contain errors
2 protocols using mouse β actin
1
Quantifying Gene Expression in Mouse Colon
2
Quantitative Real-Time PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from cells using TRIzol® reagent (cat. no. CW0580S; CoWin Biosciences) according to the manufacturer's instructions. Total RNA was quantified using a micro-spectrophotometer (NAno-300; cat. no. MO00040009; Hangzhou Allsheng Instruments Co., Ltd.), and the optical density 260/280 nm ratio of all samples was 1.8–2.0. Next, cDNA was synthesized on a PCR instrument (SimpliAmp™; cat. no. A24811; Thermo Fisher Scientific, Inc.) using the RNA reverse transcription kit (cat. no. CW2569M; CoWin Biosciences). RT was performed as follows: 42°C for 15 min, incubate at 85°C for 5 min, and keep warm at 4°C. Primers and a fluorescent quantitative PCR kit (cat. no. CW2601H; CoWin Biosciences) containing SYBR Green I fluorescent dye were used to perform fluorescent qPCR on a PCR system (cat. no. 4351106; Thermo Fisher Scientific, Inc.). Primer sequences for human HK2, human GAPDH, mouse HK2 and mouse β-actin (Sangon Biotech Co., Ltd.) are listed in Table II . The thermocycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. The 2−ΔΔCq method was used for analysis of results and gene levels were normalized to the internal reference gene, GAPDH for human HK2 or β-actin for mouse HK2 (27 (link)).
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!